Comparative Proteomic Analyses of <Emphasis Type="Italic">Streptococcus suis</Emphasis> Serotype 2 Cell Wall-Associated Proteins

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen that is distributed throughout the world. Virulence factors and/or markers of the virulent serotype 2 strains have not been fully identified. In this study a simple, rapid, and non-destructive method was used to extract cell wall-associated proteins from SS2 strains. Two virulent strains were compared with one avirulent strain by 2-dimensional electrophoresis (2DE). When the results of the 2DE analyses were combined with the results of mass spectrometry analyses, a total of 40 unique proteins were identified, including 26 antigens (2DE immunoblotting was used as a preliminary study). In addition to a known virulence factor, muramidase-released protein, two new proteins, catabolite control protein A and leucyl aminopeptidase, and nine potential virulence factors were also identified. The formers may be a potential virulence regulator or drug target, and the latter contains plasminogen-binding proteins and molecular chaperones. Our results complemented previous immunoproteomics studies of SS2 strains.     

A proteomic approach analysing the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> response to virulent and avirulent <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> strains

The present work is directed at studying changes at the proteome level in Arabidopsis thaliana leaves in response to Pseudomonas syringae virulent (Pst) and avirulent (Pst avrRpt2) strains. Arabidopsis leaves were sampled from challenged plants at 4, 8 and 24 h post inoculation. Proteins were TCA–acetone–phenol extracted and subjected to 2-DE (5–8 pH range) and MS/MS (MALDI–TOF–TOF) analysis. Out of 800 matched spots on each of the 36 gels analysed, 147 spots were either absent in at least one of the conditions studied (time or treatments; qualitative variable spots) or differentially accumulated between time and treatments (quantitative variable spots). Out of the 24 proteins successfully identified over TAIR10 database, 23 have not been reported previously in similar proteomics studies of the Arabidopsis thaliana–Pseudomonas syringae interaction. The exhaustive statistical analysis performed, including principal component and heat map, showed that 24 h post inoculation can clearly discriminate the challenged plants from the control. The protein change occurred early (4 h post inoculation) following the virulent pathogen infection, whereas the change occurred later (24 h post inoculation) following the avirulent pathogen inoculation. Concerning the variable proteins, three behavioural groups can be observed: group 1 (common protein changes in response to virulent and avirulent pathogen infection), group 2 (protein changes in response to virulent pathogen infection) and group 3 (protein changes in response to avirulent pathogen infection). Differential identified proteins following the pathogen infection belonged to different groups including those of oxidative stress defence, enzymes of metabolic pathways and molecular chaperones.     

猪链球菌2型可能的毒力基因的发现

猪链球菌2型(SS2)感染已成为影响全世界养猪业的重要问题之一。SS2菌株可分为毒力株、弱毒力株和无毒力株,但目前尚无区分此3类菌株的快速、有效的检测方法。为了获得毒力株特异的基因序列,对毒力株HA9801及无毒力株12^#进行了抑制性差减杂交(SSH)实验,获得了5个可能的新的毒力基因片段,分别是转录调节子、氨基酸通透酶、ABC转运子及表面锚定蛋白,在国内外尚属首次报道。这一发现将有助于区分SS2型菌株的毒力类型,并为SS2毒力株检测方法的建立奠定基础。     

鸭疫里默氏杆菌强、弱菌株外膜蛋白的比较蛋白质组学研究

应用双向电泳及质谱技术对血清2型鸭疫里默氏杆菌强毒株及其体外传代200代(RA200)的弱毒菌株的外膜蛋白进行比较蛋白质组学研究,借此分析鸭疫里默氏杆菌的外膜蛋白表达特点,研究差异表达蛋白与细菌毒力的关系.在实验中检测到血清2型鸭疫里默氏杆菌原代及其体外传代获得的弱毒菌株的外膜蛋白约表达60个蛋白质点(n=3),其中相差5倍以上3个.胶内酶解和肽质量指纹图谱分析后鉴定,W1为热休克蛋白Hsp20家族成员,W2、W3为转座酶,推测它们可能与里默氏杆菌的毒力密切相关.     

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.     

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.     

Differential proteomic analysis of the Bacillus anthracis secretome: distinct plasmid and chromosome CO2-dependent cross talk mechanisms modulate extracellular proteolytic activities

The secretomes of a virulent Bacillus anthracis strain and of avirulent strains (cured of the virulence plasmids pXO1 and pXO2), cultured in rich and minimal media, were studied by a comparative proteomic approach. More than 400 protein spots, representing the products of 64 genes, were identified, and a unique pattern of protein relative abundance with respect to the presence of the virulence plasmids was revealed. In minimal medium under high CO(2) tension, conditions considered to simulate those encountered in the host, the presence of the plasmids leads to enhanced expression of 12 chromosome-carried genes (10 of which could not be detected in the absence of the plasmids) in addition to expression of 5 pXO1-encoded proteins. Furthermore, under these conditions, the presence of the pXO1 and pXO2 plasmids leads to the repression of 14 chromosomal genes. On the other hand, in minimal aerobic medium not supplemented with CO(2), the virulent and avirulent B. anthracis strains manifest very similar protein signatures, and most strikingly, two proteins (the metalloproteases InhA1 and NprB, orthologs of gene products attributed to the Bacillus cereus group PlcR regulon) represent over 90% of the total secretome. Interestingly, of the 64 identified gene products, at least 31 harbor features characteristic of virulence determinants (such as toxins, proteases, nucleotidases, sulfatases, transporters, and detoxification factors), 22 of which are differentially regulated in a plasmid-dependent manner. The nature and the expression patterns of proteins in the various secretomes suggest that distinct CO(2)-responsive chromosome- and plasmid-encoded regulatory factors modulate the secretion of potential novel virulence factors, most of which are associated with extracellular proteolytic activities.     

Colonization of turbot tissues by virulent and avirulent Aeromonas salmonicida subsp. salmonicida strains during infection

Preventing disease outbreaks in cultured turbot Psetta maxima L. caused by Aeromonas salmonicida subsp. salmonicida (ASS) requires a better understanding of how this pathogen colonizes its host. Distribution of 1 virulent and 2 avirulent ASS strains in turbot tissues was investigated during early and late stages of infection following an immersion challenge. To track bacteria within the turbot, the ASS strains were tagged with green fluorescent protein (GFP). Both virulent and avirulent strains colonized the epidermal mucus, gills, and intestine within the first 12 h post challenge, suggesting that these sites may serve as points of entry into turbot. Although the avirulent strains colonized these initial sites in the turbot tissues, they were rarely found in the internal organs and were cleared from the host 4 d post challenge. In contrast, the virulent ASS strain was found in the liver and kidney as early as 12 h post challenge and was found in the muscle tissue at very late stages of infection. The virulent strain persisted in all tested host tissues until death occurred 7 d post challenge, suggesting that ASS must colonize and survive within the turbot tissues for an infection to result in death of the fish. Comparisons of the distribution profiles of both virulent and avirulent strains during early and late stages of an infection in turbot has provided important information on the route and persistence of an ASS infection in this host.     

Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene.

We developed a model system to study the signal transduction pathways leading to the activation of Arabidopsis thaliana genes involved in the defense against pathogen attack. Here we describe the identification and characterization of virulent and avirulent Pseudomonas syringae strains that elicit disease or resistance symptoms when infiltrated into Arabidopsis leaves. The virulent and avirulent strains were characterized by determining growth of the pathogen in Arabidopsis leaves and by measuring accumulation of mRNA corresponding to Arabidopsis phenylalanine ammonia-lyase (PAL), beta-1,3-glucanase (BG), and chalcone synthase (CHS) genes in infected leaves. The virulent strain, P. syringae pv maculicola ES4326, multiplied 10(5)-fold in Arabidopsis leaves and strongly elicited BG1, BG2, and BG3 mRNA accumulation but had only a modest effect on PAL mRNA accumulation. In contrast, the avirulent strain, P. syringae pv tomato MM1065, multiplied less than 10-fold in leaves and had only a minimal effect on BG1, BG2, and BG3 mRNA accumulation, but it induced PAL mRNA accumulation. No accumulation of CHS mRNA was found with either ES4326 or MM1065. We also describe the cloning of a putative avirulence (avr) gene from the avirulent strain MM1065 that caused the virulent strain ES4326 to grow less well in leaves and to strongly elicit PAL but not BG1 and BG3 mRNA accumulation. These results suggest that the Arabidopsis PAL and BG genes may be activated by distinct signal transduction pathways and show that differences in plant gene induction by virulent and avirulent strains can be attributed to a cloned presumptive avr gene.     

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 2,138,568 bp 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 105 K genomic island in strain SC070731 that is absent in seven other sequenced SS2 strains. Further analysis of the 105 K 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 105 K 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.     

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.     

Comparative proteome analysis of cellular proteins extracted from highly virulent Francisella tularensis ssp. tularensis and less virulent F. tularensis ssp. holarctica and F. tularensis ssp. mediaasiatica

Francisella tularensis is the causative agent of the zoonotic disease tularemia. Four subspecies of this pathogen, namely ssp. tularensis, mediaasiatica, holarctica, and novicida are spread throughout the northern hemisphere. Although there are marked variations in their virulence to mammals, the subspecies are difficult to identify as they are closely genetically related. We carried out the comparative proteome analysis of cellular extracts from isolates representing the highly virulent subspecies tularensis, and the less virulent subspecies mediaasiatica and holarctica in order to identify new diagnostic markers and putative factors of virulence. We identified 27 protein spots that were either specifically present or at significantly higher abundance in ssp. tularensis strains, 22 proteins in ssp. mediaasiatica strains, and 26 proteins in ssp. holarctica strains. Subspecies tularensis-specific proteins might represent putative virulence factors. Of 27 identified tularensis-specific spots 17 represented charge and mass variants of proteins occurring in other subspecies, 7 spots were found to be present at higher abundance, and 3 spots were specifically present in tularensis strains. Amongst them, PilP protein, as a component necessary for the biogenesis of the type IV pilus, virulence and adhesion factor for many human pathogen, was identified. Furthermore, the identification of additional 27 proteins common for ssp. tularensis and mediaasiatica, and 19 proteins shared by ssp. mediaasiatica and holarctica documented apparent closer genetic similarity between ssp. tularensis and mediaasiatica.     

Development of a Listeria monocytogenes EGDe partial proteome reference map and comparison with the protein profiles of food isolates

A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism. The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes. In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed. The method used provided partial fractionation of membrane and cytosolic proteins. The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L. monocytogenes EGDe proteome. An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome. The variation was greater for the less intense spots, and on average 28% of these spots were not matched. Two of the proteins identified in L. monocytogenes EGDe were missing in one or more of the food isolates. These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups. The two corresponding genes were found by PCR amplification to be present in the four food isolates. Our results show that the L. monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen.     

Comparative proteome analysis of fractions enriched for membrane-associated proteins from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica strains

The facultative intracellular pathogen Francisella tularensis is the causative agent of the serious infectious disease tularemia. Despite intensive research, the virulence factors and pathogenetic mechanisms remain largely unknown. To identify novel putative virulence factors, we carried out a comparative proteome analysis of fractions enriched for membrane-associated proteins isolated from the highly virulent subspecies tularensis strain SCHU S4 and three representatives of subspecies holarctica of different virulence including the live vaccine strain. We identified six proteins uniquely expressed and four proteins expressed at significantly higher levels by SCHU S4 compared to the ssp. holarctica strains. Four other protein spots represented mass and charge variants and seven spots were charge variants of proteins occurring in the ssp. holarctica strains. The genes encoding proteins of particular interest were examined by sequencing in order to confirm and explain the findings of the proteome analysis. Our studies suggest that the subspecies tularensis-specific proteins represent novel potential virulence factors.     

Structural and nonstructural protein genome regions of eastern equine encephalitis virus are determinants of interferon sensitivity and murine virulence

Eastern equine encephalitis virus (EEEV) causes sporadic epidemics of human and equine disease in North America, but South American strains have seldom been associated with human neurologic disease or mortality, despite serological evidence of infection. In mice, most North American and South American strains of EEEV produce neurologic disease that resembles that associated with human and equine infections. We identified a South American strain that is unable to replicate efficiently in the brain or cause fatal disease in mice yet produces 10-fold higher viremia than virulent EEEV strains. The avirulent South American strain was also sensitive to human interferon (IFN)-alpha, -beta, and -gamma, like most South American strains, in contrast to North American strains that were highly resistant. To identify genes associated with IFN sensitivity and virulence, infectious cDNA clones of a virulent North American strain and the avirulent South American strain were constructed. Two reciprocal chimeric viruses containing swapped structural and nonstructural protein gene regions of the North American and South American strains were also constructed and found to replicate efficiently in vitro. Both chimeras produced fatal disease in mice, similar to that caused by the virulent North American strain. Both chimeric viruses also exhibited intermediate sensitivity to human IFN-alpha, -beta, and -gamma compared to that of the North American and South American strains. Virulence 50% lethal dose assays and serial sacrifice experiments further demonstrated that both structural and nonstructural proteins are important contributors to neurovirulence and viral tissue tropism. Together, the results of this study emphasize the complex and important influences of structural and nonstructural protein gene regions on EEEV virulence.     

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.     

Mapping and comprehensive analysis of the extracellular and cell surface proteome of the human pathogen Corynebacterium diphtheriae

Secreted proteins of the human pathogen Corynebacterium diphtheriae might be involved in important pathogen-host cell interactions. Here, we present the first systematic reference map of the extracellular and cell surface proteome fractions of the type strain C. diphtheriae C7s(-)tox-. The analysis window of 2-DE covered the pI range from 3 to 10 along with a MW range from 8 to 150 kDa. Computational analysis of the 2-D gels detected almost 150 protein spots in the extracellular proteome fraction and about 80 protein spots of the cell surface proteome. MALDI-TOF-MS and PMF with trypsin unambiguously identified 107 extracellular protein spots and 53 protein spots of the cell surface, representing in total 85 different proteins of C. diphtheriae C7s(-)tox-. Several of the identified proteins are encoded by pathogenicity islands and might represent virulence factors of C. diphtheriae. Additionally, four solute-binding proteins (HmuT, Irp6A, CiuA, and FrgD) of different iron ABC transporters were identified, with the hitherto uncharacterized FrgD protein being the most abundant one of the cell surface proteome of C. diphtheriae C7s(-)tox-.     

Increase of capsular material thickness following in vivo growth of virulent Streptococcus suis serotype 2 strains

Abstract Protein profile and capsular material thickness of Streptococcus suis serotype 2 strains were compared after in vitro and in vivo growth. Three virulent and one avirulent strains were used. These strains were grown in Brain Heart Infusion (BHI) broth, cells were collected by centrifugation, resuspended in a sterile saline solution and injected in diffusion chambers. The devices were then inserted in rat abdomens for 17 h. In vitro grown strains were also inoculated into fresh BHI broth and cultivated for 17 h at 37°C. In vivo as well as in vitro grown bacteria were harvested by centrifugation, processed in a French pressure cell, treated with lysozyme and centrifuged to collect cell proteins for SDS-PAGE analysis. Transmission electron microscopy using polycationic ferritin labeling to stabilize capsular material was also carried out. No significant modification was noted in the protein profile for any strain after in vivo growth except for a 39 kDa protein of one virulent strain. On the other hand, an increase in thickness of capsular material was noted for the three in vivo grown virulent strains while no change was noted for the avirulent strain. This increase in capsular material thickness of virulent strains was accompanied by an increased resistance to killing by pig polymorphonuclear leukocytes. The capacity to produce more capsular material in vivo seems to be an attribute of some virulent S. suis serotype 2 strains.     

Virulent and Avirulent Strains of Babesia bovis: The Relationship Between Parasite Protease Content and Pathophysiological Effect of the Strain

A virulent strain of Babesia bovis (“L” strain) was rendered avirulent by irradiation with 35 krads with a γ source. Another virulent strain of B. bovis (“C” strain) was made avirulent by rapid blood passage through 12 splenectomised calves. Both the parent virulent and their respective avirulent strains were injected into susceptible cattle. A nonfatal disease was observed in those intact cattle that had avirulent parasites; however, a fatal disease was produced in those animals that had received virulent parasites and in splenectomised calves that had received avirulent parasites. Blood kinin levels rose and plasma kininogen levels fell significantly in those animals infected with both virulent strains. Nonsignificant changes occurred with these parameters in animals infected with avirulent parasites. Preparations of disrupted parasites were obtained from the four parasite populations. Both virulent strains contained high levels of protease. The avirulent forms contained insignificant amounts. As parasite doubling times and maximum parasitaemias were the same for all four parasite populations, we conclude that these enzymes are not obligatory for parasite multiplication in the vertebrate host. Their role in producing pathological changes in the host is discussed.