Zongfu Wu

Functional analysis of luxS in Streptococcus suis reveals a key role in biofilm formation and virulence.

Streptococcus suis (SS) is an important pathogen of pigs, responsible for diverse diseases in swine and human. LuxS 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 was constructed using homologous recombination and its biofilm formation, hemolytic activity, cell adherence, virulence and expression of virulence factors were evaluated. Compared to the parental strain, the biofilm formation and hemolytic activity were significantly decreased in the luxS mutant. The addition of synthetic autoinducer 2 could 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 strain. 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. These results suggest that luxS may be involved in the interruption of bacterial communication and biofilm formation that contribute to the virulence of the bacterium.

Immunoproteomic assay of surface proteins of Streptococcus suis serotype 9.

Streptococcus suis is an important swine pathogen responsible for a diverse group of diseases. Studies on vaccines have focused on S. suis serotype 2 strains, which are the most invasive isolates worldwide. However, in China S. suis serotype 9 (SS9) is also a prevalent serotype, which is frequently isolated from diseased pigs. Little is known about immunogenic proteins for SS9. Therefore, an immunoproteomic-based approach was developed to identify immunogenic proteins of SS9. Cell wall proteins extracted from SS9 strain GZ0565 isolated from a diseased pig with meningitis were screened by two-dimensional Western blotting using anti-SS9 sera pooled from specific pathogen-free mice. Protein spots were excised from preparative gels and identified by matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF-MS) or MALDI-TOF-TOF-MS, which led to the identification of eight immunogenic proteins (arginine deiminase, extracellular solute-binding protein, translation elongation factor Ts, neprilysin, peptide ATP-binding cassette transporter peptide-binding protein, pyruvate kinase, phosphate acetyltransferase, and fructose-bisphosphate aldolase). These immunogenic proteins, which are encoded by genes that are reasonably conserved among SS9 strains, could be developed as vaccine candidates.

Transcriptome profiling of zebrafish infected with Streptococcus suis

Streptococcus suis is an important pathogen in swine, and it also represents an emerging zoonotic agent. Zebrafish as a model for the evaluation of virulence of S. suis has been demonstrated before. Here, an Affymetrix Zebrafish GeneChip was used to identify alterations in gene expression of zebrafish injected with S. suis serotype 2 strain HA9801. The results showed that 189 genes were differentially expressed, of which 125 genes were upregulated and 64 genes were downregulated. Gene Ontology category and KEGG pathway were analyzed for differentially expressed genes. Upregulated genes were involved in response to bacterium, immune response, inflammatory response, complement activation, defense response. Three genes (encoding serum amyloid protein A, matrix metalloproteinase 9 and apoptosis-related cysteine protease) and genes involved in the regulation of IL-6 biosynthetic process, which have previously been implicated in the response to S. suis infection in other organisms, were also upregulated. Downregulated genes played roles in glycolysis, carbohydrate metabolic process, amino acids metabolism, behavior and muscle. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR on 12 representative genes. The data may provide further validation of this model, which will contribute to understanding of S. suis pathogenic mechanisms.

Reduced virulence is an important characteristic of biofilm infection of Streptococcus suis.

Streptococcus 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 with SS2 avirulent strain T15. In addition, a 58% reduction in adherence to HEp-2 cells was observed for HA9801 biofilm cells, compared with HA9801 planktonic cells. The 50% lethal dose (LD(50) ) 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 LD(50) 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 a novel insight into bacterium infection mechanisms, which suggests that a virulent strain may be able to decrease its virulence by forming a biofilm so that it can achieve persistent infection in vivo.

Comparative Proteomic Analysis of Streptococcus suis Biofilms and Planktonic Cells That Identified Biofilm Infection-Related Immunogenic Proteins

Streptococcus suis (SS) is a zoonotic pathogen that causes severe disease symptoms in pigs and humans. Biofilms of SS bind to extracellular matrix proteins in both endothelial and epithelial cells and cause persistent infections. In this study, the differences in the protein expression profiles of SS grown either as planktonic cells or biofilms were identified using comparative proteomic analysis. The results revealed the existence of 13 proteins of varying amounts, among which six were upregulated and seven were downregulated in the Streptococcus biofilm compared with the planktonic controls. The convalescent serum from mini-pig, challenged with SS, was applied in a Western blot assay to visualize 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/TOF-MS. Of these nine proteins, five (Manganese-dependent superoxide dismutase, UDP-N-acetylglucosamine 1-carboxyvinyltransferase, ornithine carbamoyltransferase, phosphoglycerate kinase, Hypothetical protein SSU05_0403) had no previously reported immunogenic properties in SS to our knowledge. The remaining four immunogenic proteins (glyceraldehyde-3-phosphate dehydrogenase, hemolysin, pyruvate dehydrogenase and DnaK) were identified under both planktonic and biofilm growth conditions. In conclusion, the protein expression pattern of SS, grown as biofilm, was different from the SS grown as planktonic cells. These five immunogenic proteins that were specific to SS biofilm cells may potentially be targeted as vaccine candidates to protect against SS biofilm infections. The four proteins common to both biofilm and planktonic cells can be targeted as vaccine candidates to protect against both biofilm and acute infections.

The Streptococcus suis transcriptional landscape reveals adaptation mechanisms in pig blood and cerebrospinal fluid

Streptococcus suis (SS) is an important pathogen of pigs, and it is also recognized as a zoonotic agent for humans. SS infection may result in septicemia or meningitis in the host. However, little is known about genes that contribute to the virulence process and survival within host blood or cerebrospinal fluid (CSF). Small RNAs (sRNA) have emerged as key regulators of virulence in several bacteria, but they have not been investigated in SS. Here, using a differential RNA-sequencing approach and RNAs from SS strain P1/7 grown in rich medium, pig blood, or CSF, we present the SS genome-wide map of 793 transcriptional start sites and 370 operons. In addition to identifying 29 sRNAs, we show that five sRNA deletion mutants attenuate SS virulence in a zebrafish infection model. Homology searches revealed that 10 sRNAs were predicted to be present in other pathogenic Streptococcus species. Compared with wild-type strain P1/7, sRNAs rss03, rss05, and rss06 deletion mutants were significantly more sensitive to killing by pig blood. It is possible that rss06 contributes to SS virulence by indirectly activating expression of SSU0308, a virulence gene encoding a zinc-binding lipoprotein. In blood, genes involved in the synthesis of capsular polysaccharide (CPS) and subversion of host defenses were up-regulated. In contrast, in CSF, genes for CPS synthesis were down-regulated. Our study is the first analysis of SS sRNAs involved in virulence and has both improved our understanding of SS pathogenesis and increased the number of sRNAs known to play definitive roles in bacterial virulence.

Comparative genomic analysis shows that Streptococcus suis meningitis isolate SC070731 contains a unique 105 K genomic island

Streptococcus suis (SS) is an important swine pathogen worldwide that occasionally causes serious infections in humans. SS infection may result in meningitis in pigs and humans. The pathogenic mechanisms of SS are poorly understood. Here, we provide the complete genome sequence of S. suis serotype 2 (SS2) strain SC070731 isolated from a pig with meningitis. The chromosome is 2,138,568bp in length. There are 1933 predicted protein coding sequences and 96.7% (57/59) of the known virulence-associated genes are present in the genome. Strain SC070731 showed similar virulence with SS2 virulent strains HA9801 and ZY05719, but was more virulent than SS2 virulent strain P1/7 in the zebrafish infection model. Comparative genomic analysis revealed a unique 105K genomic island in strain SC070731 that is absent in seven other sequenced SS2 strains. Further analysis of the 105K genomic island indicated that it contained a complete nisin locus similar to the nisin U locus in S. uberis strain 42, a prophage similar to S. oralis phage PH10 and several antibiotic resistance genes. Several proteins in the 105K genomic island, including nisin and RelBE toxin-antitoxin system, contribute to the bacterial fitness and virulence in other pathogenic bacteria. Further investigation of newly identified gene products, including four putative new virulence-associated surface proteins, will improve our understanding of SS pathogenesis.

Streptococcus suis small RNA rss04 contributes to the induction of meningitis by regulating capsule synthesis and by inducing biofilm formation in a mouse infection model

Streptococcus suis (SS) is an important pathogen for pigs, and it is also considered as a zoonotic agent for humans. Meningitis is one of the most common features of the infection caused by SS, but little is known about the mechanisms of SS meningitis. Recent studies have revealed that small RNAs (sRNAs) have emerged as key regulators of the virulence in several bacteria. In the previous study, we reported that SS sRNA rss04 was up-regulated in pig cerebrospinal fluid and contributes to SS virulence in a zebrafish infection model. Here, we show that rss04 facilitates SS invasion of mouse brain and lung in vivo. Label-free quantitation mass spectrometry analysis revealed that rss04 regulates transcriptional regulator CcpA and several virulence factors including LuxS. Transmission electron microscope and Dot-blot analyses indicated that rss04 represses capsular polysaccharide (CPS) production, which in turn facilitates SS adherence and invasion of mouse brain microvascular endothelial cells bEnd.3 in vitro and activates the mRNA expression of TLR2, CCL2, IL-6 and TNF-α in mouse brain in vivo at 12h post-infection. In addition, rss04 positively regulates SS biofilm formation. Survival analysis of infected mice showed that biofilm state in brain contributes to SS virulence by intracranial subarachnoidal route of infection. Together, our data reveal that SS sRNA rss04 contributes to the induction of meningitis by regulating the CPS synthesis and by inducing biofilm formation, thereby increasing the virulence in a mouse infection model. To our knowledge, rss04 represents the first bacterial sRNA that plays definitive roles in bacterial meningitis.

Immunoproteomic analysis of bacterial proteins of Actinobacillus pleuropneumoniae serotype 1

BackgroundActinobacillus pleuropneumoniae (APP) is one of the most important swine pathogens worldwide. Identification and characterization of novel antigenic APP vaccine candidates are underway. In the present study, we use an immunoproteomic approach to identify APP protein antigens that may elicit an immune response in serotype 1 naturally infected swine and serotype 1 virulent strain S259-immunized rabbits.ResultsProteins from total cell lysates of serotype 1 APP were separated by two-dimensional electrophoresis (2DE). Western blot analysis revealed 21 immunoreactive protein spots separated in the pH 4-7 range and 4 spots in the pH 7-11 range with the convalescent sera from swine; we found 5 immunoreactive protein spots that separated in the pH 4-7 range and 2 in the pH 7-11 range with hyperimmune sera from S259-immunized rabbits. The proteins included the known antigens ApxIIA, protective surface antigen D15, outer membrane proteins P5, subunit NqrA. The remaining antigens are being reported as immunoreactive proteins in APP for the first time, to our knowledge.ConclusionsWe identified a total of 42 immunoreactive proteins of the APP serotype 1 virulent strain S259 which represented 32 different proteins, including some novel immunoreactive factors which could be researched as vaccine candidates.

A Streptococcus suis LysM domain surface protein contributes to bacterial virulence

Streptococcus suis (SS) is a major swine pathogen, as well as a zoonotic agent for humans. Numerous factors contribute to SS virulence, but the pathogenesis of SS infection is poorly understood. Here, we show that a novel SS surface protein containing a LysM at the N-terminus (SS9-LysM) contributes to SS virulence. Homology analysis revealed that the amino acid sequence of SS9-LysM from the SS strain GZ0565 shares 99.8-68.7% identity with homologous proteins from other SS strains and 41.2% identity with Group B Streptococcal protective antigen Sip. Immunization experiments showed that 7 out of 30 mice immunized with recombinant SS9-LysM were protected against challenge with the virulent GZ0565 strain, while all of the control mice died within 48h following bacterial challenge. In mouse infection model, the virulence of the SS9-LysM deletion mutant (ΔSS9-LysM) was reduced compared with the wild-type (WT) strain GZ0565 and SS9-LysM complemented strain. In addition, ΔSS9-LysM was significantly more sensitive to killing by pig blood ex vivo and mouse blood in vivo compared with the WT strain and SS9-LysM complemented strain. In vivo transcriptome analysis in mouse blood showed that the WT strain reduced the expression of host genes related to iron-binding by SS9-LysM. Moreover, the total free iron concentration in blood from infected mice was significantly lower for the ΔSS9-LysM strain compared with the WT strain. Together, our data reveal that SS9-LysM facilitates SS survival within blood by releasing more free iron from the host. This represents a new mechanism of SS pathogenesis.