Immunoproteomic assay of secreted proteins of <Emphasis Type="Italic">Streptococcus suis</Emphasis> serotype 9 with convalescent sera from pigs

Streptococcus suis is an important pathogen of pigs. In China, in addition to S. suis serotype 2, S. suis serotype 9 (SS9) is also a prevalent serotype. There is no vaccine available for SS9. An immunoproteome-based approach was developed to identify SS9 immunogenic proteins for vaccine development. Secreted proteins extracted from SS9 strain GZ0565 were screened by two-dimensional Western blotting using convalescent sera from pigs. Protein spots were excised from preparative gels and were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, which led to the identification of ten immunogenic proteins (sortases, ABC transporter substrate-binding protein–maltose/maltodextrin, ABC transporter periplasmic protein, CHAP domain containing protein, peptidoglycan-binding LysM, elongation factor Tu, elongation factor G, thymidine kinase, molecular chaperone DnaK, hypothetical protein SSU98_2184). These novel immunogenic proteins, which are encoded by genes that are reasonably conserved among SS9 strains, may be developed as antigens for further study of SS9 vaccine.     

Differences in the population structure of invasive Streptococcus suis strains isolated from pigs and from humans in The Netherlands

Streptococcus suis serotype 2 is the main cause of zoonotic S. suis infection despite the fact that other serotypes are frequently isolated from diseased pigs. Studies comparing concurrent invasive human and pig isolates from a single geographical location are lacking. We compared the population structures of invasive S. suis strains isolated between 1986 and 2008 from human patients (N?=?24) and from pigs with invasive disease (N?=?124) in The Netherlands by serotyping and multi locus sequence typing (MLST). Fifty-six percent of pig isolates were of serotype 9 belonging to 15 clonal complexes (CCs) or singleton sequence types (ST). In contrast, all human isolates were of serotype 2 and belonged to two non-overlapping clonal complexes CC1 (58%) and CC20 (42%). The proportion of serotype 2 isolates among S. suis strains isolated from humans was significantly higher than among strains isolated from pigs (24/24 vs. 29/124; P<0.0001). This difference remained significant when only strains within CC1 and CC20 were considered (24/24 vs. 27/37,P?=?0.004). The Simpson diversity index of the S. suis population isolated from humans (0.598) was smaller than of the population isolated from pigs (0.765, P?=?0.05) indicating that the S. suis population isolated from infected pigs was more diverse than the S. suis population isolated from human patients. S. suis serotype 2 strains of CC20 were all negative in a PCR for detection of genes encoding extracellular protein factor (EF) variants. These data indicate that the polysaccharide capsule is an important correlate of human S. suis infection, irrespective of the ST and EF encoding gene type of S. suis strains.     

Identification of immunogenic cell wall-associated proteins of Streptococcus suis serotype 2

Streptococcus suis serotype 2 (SS2) is a porcine and human pathogen with adhesive and invasive properties. The absence of suitable vaccine or virulent marker can be the bottleneck to control SS2 infection. An immunoproteome-based approach was developed to identify candidate antigens of SS2 for vaccine development. Hyperimmune sera, convalescent sera, and control sera were analyzed for reactivity by Western Blot against SS2 cell wall-associated proteins (WAPs) separated by 2-DE. A total of 34 proteins were identified by immunoproteomic analysis, of which 15 were recognized by both hyperimmune sera and convalescent sera, including most WAPs currently characterized as SS2 vaccine candidate antigens: muramidase-released protein (MRP), surface protein SP1 (Sao), and glyceraldehyde-3-phosphate dehydrogenase (GapdH). The novel immunogenic proteins may be developed as alternative antigens for further study of SS2 vaccine and diagnostics.     

四川省资阳市爆发的猪链球菌感染疫情中病原菌的分离和鉴定

对四川资阳地区病猪标本、尸检肝标本和血清标本进行病原菌分离,得到10株分离菌。经菌落形态和菌体形态观察、生化鉴定,证明其中3株为猪链球菌2型(SS2)。通过对3株SS2胞外因子基因、溶菌酶释放蛋白基因、荚膜多糖基因和16S rRNA基因进行PCR扩增,结果分别在626bp8、85bp4、87bp和297bp处出现目的条带。为进一步了解分离的SS2菌株的特性,使用VITEK GPS-107药敏卡进行药敏试验,结果表明分离菌对青霉素-G、红霉素、万古霉素等多种抗生素敏感。分离菌感染Balb/c小鼠可引起动物死亡,并出现胃肠肿胀、嘴部青紫以及皮下紫斑等症状,与患者症状相似,小鼠脏器压片经革兰氏染色镜下观察可见阳性球菌。     

猪链球菌IgG结合蛋白的原核表达及其与不同动物IgG的结合性

猪链球菌IgG结合蛋白(SPG)是一种可与多种动物IgG结合的细胞壁蛋白,广泛地存在于猪链球菌的各个血清型中,被认为是共同抗原。然而其在猪链球菌中的生物学意义并不清楚。本实验用PCR方法从猪链球菌1/2、1、2和9型菌株基因组中扩增SPG基因,构建pET28a-SPG重组表达载体,将其转入大肠杆菌BL21菌株。IPTG诱导表达后,重组蛋白经SDS-PAGE及Western blotting鉴定为可高效表达。镍亲和层析及分子筛两步纯化后获得纯度较高的目的蛋白。Western blotting及ELISA试验结果表明,所有纯化的目的蛋白均可与不同动物IgG结合,其中与人和猪IgG的结合能力相对较高,但不同血清型猪链球菌SPG与同种动物IgG的结合活性没有明显差异。     

Pre-absorbed immunoproteomics: a novel method for the detection of Streptococcus suis surface proteins

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen that can cause infections in pigs and humans. Bacterial surface proteins are often investigated as potential vaccine candidates and biomarkers of virulence. In this study, a novel method for identifying bacterial surface proteins is presented, which combines immunoproteomic and immunoserologic techniques. Critical to the success of this new method is an improved procedure for generating two-dimensional electrophoresis gel profiles of S. suis proteins. The S. suis surface proteins identified in this study include muramidase-released protein precursor (MRP) and an ABC transporter protein, while MRP is thought to be one of the main virulence factors in SS2 located on the bacterial surface. Herein, we demonstrate that the ABC transporter protein can bind to HEp-2 cells, which strongly suggests that this protein is located on the bacterial cell surface and may be involved in pathogenesis. An immunofluorescence assay confirmed that the ABC transporter is localized to the bacterial outer surface. This new method may prove to be a useful tool for identifying surface proteins, and aid in the development of new vaccine subunits and disease diagnostics.     

Slaughterhouse pigs are a major reservoir of Streptococcus suis serotype 2 capable of causing human infection in southern Vietnam

Streptococcus suis is a pathogen of major economic significance to the swine industry and is increasingly recognized as an emerging zoonotic agent in Asia. In Vietnam, S. suis is the leading cause of bacterial meningitis in adult humans. Zoonotic transmission is most frequently associated with serotype 2 strains and occupational exposure to pigs or consumption of infected pork. To gain insight into the role of pigs for human consumption as a reservoir for zoonotic infection in southern Vietnam, we determined the prevalence and diversity of S. suis carriage in healthy slaughterhouse pigs. Nasopharyngeal tonsils were sampled from pigs at slaughterhouses serving six provinces in southern Vietnam and Ho Chi Minh City area from September 2006 to November 2007. Samples were screened by bacterial culture. Isolates of S. suis were serotyped and characterized by multi locus sequence typing (MLST) and pulse field gel electrophoresis (PFGE). Antibiotic susceptibility profiles and associated genetic resistance determinants, and the presence of putative virulence factors were determined. 41% (222/542) of pigs carried S. suis of one or multiple serotypes. 8% (45/542) carried S. suis serotype 2 which was the most common serotype found (45/317 strains, 14%). 80% of serotype 2 strains belonged to the MLST clonal complex 1,which was previously associated with meningitis cases in Vietnam and outbreaks of severe disease in China in 1998 and 2005. These strains clustered with representative strains isolated from patients with meningitis in PFGE analysis, and showed similar antimicrobial resistance and virulence factor profiles. Slaughterhouse pigs are a major reservoir of S. suis serotype 2 capable of causing human infection in southern Vietnam. Strict hygiene at processing facilities, and health education programs addressing food safety and proper handling of pork should be encouraged.     

Identification and characterization of two temperature-induced surface-associated proteins of Streptococcus suis with high homologies to members of the Arginine Deiminase system of Streptococcus pyogenes

The present study was performed to identify stress-induced putative virulence proteins of Streptococcus suis. For this, protein expression patterns of streptococci grown at 32, 37, and 42 degrees C were compared by one- and two-dimensional gel electrophoresis. Temperature shifts from 32 and 37 to 42 degrees C induced expression of two cell wall-associated proteins with apparent molecular masses of approximately 47 and 53 kDa. Amino-terminal sequence analysis of the two proteins indicated homologies of the 47-kDa protein with an ornithine carbamoyltransferase (OCT) from Streptococcus pyogenes and of the 53-kDa protein with the streptococcal acid glycoprotein (SAGP) from S. pyogenes, an arginine deiminase (AD) recently proposed as a putative virulence factor. Cloning and sequencing the genes encoding the putative OCT and AD of S. suis, octS and adiS, respectively, revealed that they had 81.2 (octS) and 80.2% (adiS) identity with the respective genes of S. pyogenes. Both genes belong to the AD system, also found in other bacteria. Southern hybridization analysis demonstrated the presence of the adiS gene in all 42 serotype 2 and 9 S. suis strains tested. In 9 of these 42 strains, selected randomly, we confirmed expression of the AdiS protein, homologous to SAGP, by immunoblot analysis using a specific antiserum against the SAGP of S. pyogenes. In all strains AD activity was detected. Furthermore, by immunoelectron microscopy using the anti-S. pyogenes SAGP antiserum we were able to demonstrate that the AdiS protein is expressed on the streptococcal surface in association with the capsular polysaccharides but is not coexpressed with them.     

Rapid PCR test for Streptococcus suis serotype 7

Recent epidemiological studies on Streptococcus suis infections in pigs indicated that, besides serotypes 1, 2 and 9, serotype 7 is also frequently associated with diseased animals. For the latter serotype, however, no rapid and sensitive diagnostic methods are available. This hampers prevention and control programs. Here, we describe the development of a type-specific PCR test for the rapid and sensitive detection of S. suis serotype 7. The test is based on DNA sequences of capsular (cps) genes specific for serotype 7. These sequences were identified by cross-hybridization of several individual cps genes with the chromosomal DNAs of 35 different S. suis serotypes.     

Complete genome sequence of Streptococcus suis serotype 14 strain JS14

Streptococcus suis is an important zoonotic agent leading to a variety of diseases in swine and can be transmitted to human beings upon close contact. Here, we report the complete genome sequence of S. suis serotype 14 strain JS14 which was isolated from a diseased pig in Jiangsu Province, China.     

Non-encapsulated strains reveal novel insights in invasion and survival of Streptococcus suis in epithelial cells

Streptococcus suis is a porcine and human pathogen causing invasive diseases, such as meningitis or septicaemia. Host cell interactions of S. suis have been studied mainly with serotype 2 strains, but multiple capsular serotypes as well as non-typeable strains exist with diverse virulence features. At present, S. suis is considered an extracellular pathogen. However, whether or not it can also invade host cells is a matter of controversial discussions. We have assessed adherence and invasion of S. suis for HEp-2 epithelial cells by comparing 10 serotype 2 strains and four non-typeable (NT) strains. Only the NT strains and a non-encapsulated serotype 2 mutant strain, but none of the serotype 2 strains, adhered strongly and were invasive. Invasion seemed to be affected by environmental signals, as suggested from comparison of strains grown in different media. Further phenotypic and genotypic characterization revealed a high diversity among the different strains. Electron microscopic analysis of invasion of selected invasive NT strains indicated different uptake mechanisms. One strain induced large invaginations comparable to those seen in 'caveolae' mediated uptake, whereas invasion of the other strains was accompanied by formation of filipodia-like membrane protrusions. Invasion of all strains, however, was similarly susceptible to hypertonic sucrose, which inhibits receptor-mediated endocytosis. Irrespective of the uptake pathway, streptococci resided in acidified phago-lysosome like vacuoles. All strains, except one, survived intracellularly as well as extracellular acidic conditions. Survival seemed to be associated with the AdiS protein, an environmentally regulated arginine deiminase of S. suis. Concluding, invasion and survival of NT strains of S. suis in epithelial cells revealed novel evidence that S. suis exhibits a broad variety of virulence-associated features depending on genetic variation and regulation.     

A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase

Polymerase chain reaction (PCR) primers that flank a 688-bp segment within the glutamate dehydrogenase gene (gdh) of Streptococcus suis type 2 could amplify efficiently the DNA of all 306 (100%) clinical S. suis isolates tested (pigs, n=305; human, n=1) encompassing all serotypes obtained from diverse organs, and geographic origins. When DNA from other bacteria were used as templates for amplification, no product was detected indicating specificity of the primers. Multiplex PCR was developed using the gdh gene primer pair and primers that targeted the gene encoding S. suis capsular biosynthesis (cps). This strategy enabled the detection of strains belonging to serotypes 1/2, 1, 2, 7, and 9, respectively. Using the multiplex-PCR technique, 12 out of 14 (86%) isolates that were previously identified as non-typable S. suis (based on biochemical reactions and serology) gave positive PCR results of which four were positive for serotype 7, three for serotype 2, and five for S. suis strains that belong to other serotypes. Retest results of all 14 isolates by several veterinary laboratories were identical with PCR and confirmed that the two non-PCR reactive isolates belonged to strains of other streptococcal species. These results indicated that PCR improved species determination and can thus be used as a reliable species-specific molecular diagnostic reagent for the accurate identification of S. suis isolates and a serotype-specific method for the detection of strains of serotypes 1/2, 1, 2, 7, and 9, respectively. The PCR method therefore has potential clinical and epidemiological applications.     

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.     

Infection with Streptococcus Suis Serotypes 1 and 2 in the Same Diseased Pig

Streptococcus suis is an important pig pathogen which is associated with respiratory problems, meningitis and less fre-quently with a variety of other conditions(Hommez et al. 1986). S. suis type 1 causes disease mainly in 1–2 week old pigs while serotype 2 is found commoaily in 2–22 week old pigs, S. suis type 2 is a zoonosis. It can cause meningitis and septicaemia in man (Christensen & Kronvall 1985). Several other serotypes of S. suis have also been identified on the basis of the capsular poly-saccharide (Perch et al. 1983, Hommez et al. 1986). We present a case where we isolated S. suis types 1 and 2 from the brain and lungs respectively of the same diseased suckling piglet. This i/s the first reported case of S. suis types 1 and 2 in Finland.     

Identification and characterization of four proteases produced by Streptococcus suis

Streptococcus suis is an important worldwide swine pathogen. In this study, we investigated the production of proteases by S. suis serotype 2. Proteases were identified and characterized using chromogenic and fluorogenic assays and zymography. An Arg-aminopeptidase with a molecular mass of 55 kDa was found to be both cell-associated and extracellular. Cell-associated chymotrypsin-like and caseinase activities, belonging to the serine- and metalloprotease classes respectively, were also detected. Lastly, a dipeptidyl peptidase IV (DPP IV) with a molecular mass of 70 kDa was detected in both whole cells and culture supernatants of S. suis serotype 2. Arg-aminopeptidase, caseinase and DPP IV activities were detected in all strains of S. suis serotype 2 tested whereas the chymotrypsin-like activity was only detected in European virulent strains of serotype 2. The optimum pH for all four proteases was between 6 and 8, and the optimum temperature ranged from 25 to 42 degrees C. This is the first report on the production of proteases by S. suis. Further investigations will determine the possible contribution of these proteases in the pathogenicity of S. suis serotype 2.     

猪链球菌2型次黄嘌呤核苷酸脱氢酶基因的克隆与鉴定

根据GenBank猪链球菌2型(SS2)报道序列,对江苏分离株SS2-H部分测序,发现位于已知毒力基因orf2与mrp之间存在两个新的开放阅读框序列。选取可能含有抗原决定簇肽段对应的核酸序列,该阅读框(2738~3694)编码319个氨基酸残基,分子量为33.5kDa,与已知任何基因无同源性。通过InterPro、PHD、DNAstar分析阅读框,并定向克隆至pET-32α( )载体中,转化至大肠杆菌BL21,表达出分子量为48kDa的融合蛋白,蛋白免疫转印可被SS2的抗血清识别,具有免疫原性;并且含有IMP dehydrogenase结构域,催化IMP生成XMP;流式细胞仪检测该蛋白可明显影响HEp-2细胞周期。     

Identification and characterization of novel immunogenic proteins of Streptococcus suis serotype 2

Streptococcus suis, a zoonotic pathogen, caused serious outbreaks in humans with high mortality rates in the past decade. To develop safer and more effective vaccines, particularly for human protection, cell wall and extracellular proteins of S. suis serotype 2 were analyzed by an immunoproteomic approach in this study. Thirty-two proteins with high immunogenicity were identified and 22 of them were newly identified. Further analyses of 9 selected proteins revealed that (1) these 9 proteins were expressed in all tested virulent S. suis serotype 2 isolates, (2) antisera against 6 of the selected proteins efficiently killed the bacteria by opsonized phagocytosis in human blood, and (3) significantly higher levels of serum antibodies against 3 proteins were detected in both patients and infected swines. Therefore, our results suggest the 3 proteins (SSU98_0197, SSU98_1094 and SSU1664) have strong potential to be vaccine candidates.     

溶原性噬菌体在猪链球菌2型分离株中的检出和鉴定

[目的]为了研究噬菌体整合酶基因在猪链球菌2型(Streptococcus suis type 2,SS2)中的分布情况.[方法]根据噬菌体整合酶基因设计引物,建立了PCR方法,并对扩增产物进行测序.[结果]结果显示,25株SS2致病菌株均扩增出目的片段,非毒力株T15、5株其它血清型猪链球菌及兰氏C群猪源链球菌未扩增出目的片段.经丝裂霉素C诱导后,SS2致病菌株出现完全的细胞溶解,而非毒力株T15未出现溶解.SS2致病株HA9801和ZY05719诱导均产生溶原性噬菌体,分别命名为SS2-HA和SS2-ZY,电镜观察,二者均头部呈正六边形,无尾部,其核酸类型为dsDNA,可鉴定为复层噬菌体科(Tectiviridae)的成员.噬菌体SS2-HA和SS2-ZY整合酶基因序列与已报道的SS2噬菌体整合酶基因序列高度同源,显示SS2噬菌体整合酶具有较高的特异性.[结论]从SS2致病株中检出溶原性噬菌体和噬菌体整合酶基因,且噬菌体整合酶基因与SS2溶菌酶释放蛋白(mrp)等7种毒力相关基因有相关性,表明SS2的溶原性噬菌体可能与其致病性有关.     

猪链球菌2型新的感染相关因子自溶素的鉴定与分析

根据Sanger研究所公布的猪链球菌2型(SS2)P1/7株的自溶素序列,设计检测引物,取SS2我国2次流行株、其它临床分离株和参考株,及猪链球菌1型、1/2型、7型和9型,共33株,分别以其DNA为模板,PCR扩增.结果表明,SS2除无毒株T15阴性外,其他临床分离株27株(含人源2株)均阳性;其它猪链球菌为,SS7阳性,SS1、SS1/2和SS9均阴性.同时设计引物向两侧扩增,以四川流行株ZY05719和江苏流行株HA9801的DNA为模板,扩增自溶素ORF完整的编码基因,软件分析结果显示,该基因含有6个重复的"GBS_Bsp-like"域和1个"N-乙酰胞壁酰-L-丙氨酸酰胺酶"域,与SS2欧洲株有较高同源性(99.8%),但与SS2加拿大株差异较大.在DNASTAR分析所编码蛋白的抗原性的基础上,另设计引物,以ZY05719株DNA为模板,PCR扩增具有良好免疫原性的片段基因,并定向克隆至表达载体pET30a( )中,进行重组表达,SDS-PAGE和Western blot表明,所获得重组自溶素具有良好反应原性.     

Immunoproteomics of extracellular proteins of Chinese virulent strains of Streptococcus suis type 2

Streptococcus suis type 2 (SS2) is a porcine zoonotic pathogen with worldwide distribution, and lacking suitable vaccine and virulent maker were bottleneck to control this infection. An immunoproteomic assay was used to identify antigenic proteins from the total extracellular proteins of the virulent Chinese SS2 strain ZY05719. The convalescent serum of a specific pathogen free (SPF) mini-pig recognized nine protein spots on PVDF membrane. Antigenic proteins on a duplicate gel, as well as those with a similar placement of extracellular proteins from another virulent strain (HA9801) and an avirulent strain (T15) on 2-D gels, were excised and identified by MALDI-TOF-MS. PMF of the protein spots were performed using the MASCOT server. Two proteins were found in all three strains. Comparative proteomic analysis between the two virulent strains and the avirulent strain revealed nine differential proteins, eight of which were successfully identified. Genes for six of the differentially expressed proteins were found in both virulent strains, and of those were present in the avirulent stain.