| Streptococcus suis (SS) is a Gram-positive facultative anaerobic bacteria. Thirty-three serotypes (1/2, 1-31 and 33) have been described based on capsular polysaccharides (CPS).Among them, serotype 1/2, 1? 2?7?9 and 14 all can cause disease on pigs. Serotype 2 (SS2)is considered to be the most prevalent and pathogenic type. As a zoonosis, it is not only a major swine pathogen for causing a variety of diseases in pigs including septicemia,meningitis and endocarditic, but also can infect human and lead to meningitis, septicemia and even death. Two SS2 outbreaks have emerged in China and led to severe casualties. But not all SS2 strains can cause disease. So far, extensive researches about SS2 virulence factors have been developed, but its pathogenesis is still unclear. This study intends to analyze the role of differential proteins played in the mechanisms of virulence formation from three different proteomics perspectives and a new post-translational modification(PTM) view.1 Comparative genomics and proteomics analysis between a natural SS2 high virulent strain and a low virulent strainSS2 is an important zoonotic pathogen worldwide. But not all stains of SS2 can cause disease. Comparative proteomics analysis between the proteomes of a representative virulent strain of Sichuan outbreak in China, ZY05719 and a classical European low virulent strain, T15 using iTRAQ (isobaric tag for relative and absolute quantitation) was performed, for iTRAQ is suitable for analysis of complex samples. Combing the previous study of comparative genomics analysis, we found that 33 of the 148 high virulent strain unique genes were expressed. Among the 33 differential expressed proteins, based on the analysis of prediction of operon, (I/L)(P/A)XTG motif and TL(L/V)TC motif, we found sortase. Then the intact sortase family in ZY05719 genome, including five sortase C and one sortase A was analyzed. Three encoding genes of the five sortase C only existed in high virulent strain's genome and located in the same operon and shared bases between the adjacent genes. The three genes encoding sortase C were named srtC1, srtC2 and srtC3.In the downstream of the three sortase C in the genome, also in the adjacent operon, we found a potential substrate of the three sortase C, C123, by the iTRAQ results. The characteristics of C123 were in accordance with the substrate of sortase C. Two other genes of sortase C, srtC4 and srtC5, the gene expression level of which showed high level in high virulent strains analyzed by iTRAQ were also found. Also we found the potential substrate of sortase C4, C4, the gene of which located in the same operon with srtC4. Its expression in the high virulence strain is significantly higher than in the low virulence one. This is the first systematic identification of all the sortases existed in SS2, including sortase A and sortase C. And the knock out of the genes of sortase C system attenuated the virulence of the highly virulent strain.2 Comparative proteomics analysis between artificial high and low virulence mutantsComparative proteomics, which offer vast amounts of data, has been used to discover novel putative virulence factors between differential virulence bacteria. The differences of genomes exist in all sequenced SS strains, even in the same serotype, for example, the size of genome in some SS2 strains covered from 2.0 to 2.24 Mb, such as P1/7 (2.01Mb),ZY05719 (2.09Mb), SC07031 (2.14Mb) and T15 (2.24Mb)(http://www.ncbi.nlm.nih.gov/genome/genomes/199?). Furthermore, the differences will be magnified after transformation into proteins,caused by SNPs in alleles,regulation at mRNA and proteins levels and so on. These accumulating differences would affect the accuracy of the results of comparative proteomics analysis using different natural sources of bacterial strains. Fortunately, using artificial mutants with different virulence for comparative proteomics analysis can avoid these problems, because they share almost identical genetic backgrounds, except for the deleted gene. In this study, two mutants,?pepTand ?rfeA, which were generated from the same wild-type (WT) strain, ZY05719 and showed opposite virulence tendencies,were constructed. Combing two proteomics assays, two-dimensional difference gel electrophoresis (2D-DIGE) and label-free proteomics, which were suitable to analyze the samples with similar background, we identified 38 differentially abundant proteins in the mutants compared to their parent,including five known VFs of S. suis and 33 novel elements. One of the novel proteins, a putative pilus protein, named SBP2, was considered as the most promising VF, because SBP2 was not only linked with known VFs in the virulence interaction network and was proposed as located on the cell surface, but also showed enriched distribution among high virulent strains of SS. SBP2 could also bind fibronectin and laminin, two important extracellular matrix proteins of the host to function as adhesion. Thus,spb2 was identified encoding a promising virulence-associated candidate associated with the pathogensis of SS2, and a comprehensive virulence interaction network of SS was established for the first time.3 Comparative proteomics analysis of in vitro and in vivo culture of high virulent SS2Most researches of SS2 were based on study of in vitro, which could not reflect the real in vivo infection process. In this research, global proteomics changes of the bacterial proteome extracted from the isolates purified from blood of infected pigs,against those extracted from in vitro culture were analyzed. Total 188 proteins showed differential abundance,including 99 abundance-decreased and 89 increased proteins in in vivo samples against the in vitro ones,among which there were only six known virulence factors,glutamate dehydrogenase (GDH), muramidase-released protein (MRP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and so on. More interesting findings were the other novel differential proteins. For example, fatty acid biosynthesis and metabolism associated proteins showed significantly decreased abundance, while the amino acids synthesis and glucose metabolism associated proteins showed significantly increased trend. Among them,we found that one of the down-regulated fatty acid biosynthesis and metabolism associated protein,fabGs the abundance of which was reduced,the bacteria will be better able to survive in the blood. While one of the up-regulated proteins, the important hub implicated in the network consisting of known infection related virulence factors, the bifunctional acetaldehyde-CoA/alcohol dehydrogenase (adhE) was found not only responsible for anaerobic fermentation,but also associated with the bacteria pathogenicity through adhesion to Caco-2 cells with Hsp60. In sum,a rough network that responsible to sense the environment to allow the bacterium to adapt to new encountered conditions and causing infection in pigs was established for the first time.4 Succinylation and Acetylation of SS2 bacterial proteinsLysine acylation is an important post-translational modification (PTM),including acetylation and succinylation, etc., and is associated with various cellular processes. Not only the difference in protein abundance can cause the difference of virulence, the PTM of proteins is also closely related to virulence. It is reported that lysine succinylation is related to Brucella virulence and lysine acetylation contributes to the virulence of Salmonella.However, as an important zoonotic pathogen, to our knowledge, no lysine acylated protein in SS has been identified, presenting a major obstacle to understand the regulatory roles of lysine acylation in this life-threatening pathogen. In order to fill this gap, we have initiated a systematic study of the identities and functional roles of the acylated protein in SS.We analyzed the succinylome and acetylome of high virulent SS2 strain, ZY05719 by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated and acetylated peptides from digested cell lysates and subsequent peptide identification. In total,76 lysine succinylation sites on 51 proteins (2.6% of total proteins) and 2901 lysine acetylation sites on 888 proteins (46.1% of total proteins) were identified in SS. A large proportion of the succinylation and acetylation proteins are involved in various biological process and metabolic process. We further dissected the regulatory role of acetylation on transporter, adhesion, regulation and signaling. And both modifications showed extensive overlap and take place in the known virulence factors, such as MRP, GAPDH, EF-Tu,enolase and so on, which suggested the potential virulence associated function of succinylation and acetylation in SS. But the different motifs succinylation and acetylation preferred suggested that they contributed in a different way and may work together.Together, our findings revealed widespread roles for lysine succinylation and acetylation in regulating metabolism and diverse processes in SS. Our data provided a rich resource for functional analyses of lysine succinylation and acetaylation and layed foundation for further study. |
PDF Full Text Request