scholarly journals Proteomic Analysis and Identification of Streptococcus pyogenes Surface-Associated Proteins

2006 ◽  
Vol 189 (5) ◽  
pp. 1514-1522 ◽  
Author(s):  
Anatoly Severin ◽  
Elliott Nickbarg ◽  
Joseph Wooters ◽  
Shakey A. Quazi ◽  
Yury V. Matsuka ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Vaccine Development ◽  
Wide Spectrum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Surface ◽  
Cell Enzyme ◽  
Specific Antisera ◽  
Significant Burden ◽  
Associated Proteins ◽  
Surface Localization

ABSTRACT Streptococcus pyogenes is a gram-positive human pathogen that causes a wide spectrum of disease, placing a significant burden on public health. Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. The identification of these proteins for vaccine development is an important goal of bacterial proteomics. Here we describe a method of proteolytic digestion of surface-exposed proteins to identify surface antigens of S. pyogenes. Peptides generated by trypsin digestion were analyzed by multidimensional tandem mass spectrometry. This approach allowed the identification of 79 proteins on the bacterial surface, including 14 proteins containing cell wall-anchoring motifs, 12 lipoproteins, 9 secreted proteins, 22 membrane-associated proteins, 1 bacteriophage-associated protein, and 21 proteins commonly identified as cytoplasmic. Thirty-three of these proteins have not been previously identified as cell surface associated in S. pyogenes. Several proteins were expressed in Escherichia coli, and the purified proteins were used to generate specific mouse antisera for use in a whole-cell enzyme-linked immunosorbent assay. The immunoreactivity of specific antisera to some of these antigens confirmed their surface localization. The data reported here will provide guidance in the development of a novel vaccine to prevent infections caused by S. pyogenes.

scholarly journals Monoclonal Antibodies to Shigella Lipopolysaccharide Are Useful for Vaccine Production

2016 ◽  
Vol 23 (8) ◽  
pp. 681-688 ◽  
Author(s):  
Jisheng Lin ◽  
Mark A. Smith ◽  
William H. Benjamin ◽  
Robert W. Kaminski ◽  
Heather Wenzel ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Vaccine Development ◽  
Shigella Flexneri ◽  
High Sensitivity ◽  
Epidemiological Data ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Bacterial Surface ◽  
Bactericidal Assay

ABSTRACTThere is a significant need for an effective multivalentShigellavaccine that targets the most prevalent serotypes. MostShigellavaccines under development utilize serotype-specific lipopolysaccharides (LPSs) as a major component based on protection and epidemiological data. As vaccine formulations advance from monovalent to multivalent, assays and reagents need to be developed to accurately and reproducibly quantitate the amount of LPSs from multiple serotypes in the final product. To facilitate this effort, we produced 36 hybridomas that secrete monoclonal antibodies (MAbs) against the O antigen on the LPS fromShigella flexneri2a,Shigella flexneri3a, andShigella sonnei. We used six of these monoclonal antibodies for an inhibition enzyme-linked immunosorbent assay (iELISA), measuring LPSs with high sensitivity and specificity. It was also demonstrated that theShigellaserotype-specific MAbs were useful for bacterial surface staining detected by flow cytometry. These MAbs are also useful for standardizing the serum bactericidal assay (SBA) forShigella. Functional assays, such as thein vitrobactericidal assay, are necessary for vaccine evaluation and may serve as immunological correlates of immunity. AnS. flexneri2a-specific monoclonal antibody killedS. flexneri2b isolates, suggesting thatS. flexneri2a LPS may induce cross-protection againstS. flexneri2b. Overall, theShigellaLPS-specific MAbs described have potential utility to the vaccine development community for assessing multivalent vaccine composition and as a reliable control for multiple immunoassays used to assess vaccine potency.

Cysteine proteinase SpeB from Streptococcus pyogenes – a potent modifier of immunologically important host and bacterial proteins

2011 ◽  
Vol 392 (12) ◽  
pp. 1077-1088 ◽  
Author(s):  
Daniel C. Nelson ◽  
Julia Garbe ◽  
Mattias Collin
Keyword(s):  
Streptococcus Pyogenes ◽  
Group A Streptococcus ◽  
Wide Spectrum ◽  
Cysteine Proteinase ◽  
Complement Components ◽  
Bacterial Surface ◽  
Bacterial Proteins ◽  
Disease States ◽  
Group A ◽  
Important Host

AbstractGroup A streptococcus (Streptococcus pyogenes) is an exclusively human pathogen that causes a wide spectrum of diseases ranging from pharyngitis, to impetigo, to toxic shock, to necrotizing fasciitis. The diversity of these disease states necessitates thatS. pyogenespossess the ability to modulate both the innate and adaptive immune responses. SpeB, a cysteine proteinase, is the predominant secreted protein fromS. pyogenes. Because of its relatively indiscriminant specificity, this enzyme has been shown to degrade the extracellular matrix, cytokines, chemokines, complement components, immunoglobulins, and serum protease inhibitors, to name but a few of the known substrates. Additionally, SpeB regulates other streptococcal proteins by degrading them or releasing them from the bacterial surface. Despite the wealth of literature on putative SpeB functions, there remains much controversy about this enzyme because many of reported activities would produce contradictory physiological results. Here we review all known host and bacterial protein substrates for SpeB, their cleavage sites, and discuss the role of this enzyme in streptococcal pathogenesis based on the current literature.

scholarly journals Surfaceome of Leptospira spp.

2005 ◽  
Vol 73 (8) ◽  
pp. 4853-4863 ◽  
Author(s):  
Paul A. Cullen ◽  
Xiaoyi Xu ◽  
James Matsunaga ◽  
Yolanda Sanchez ◽  
Albert I. Ko ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Surface Expression ◽  
Surface Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Two Dimensional Gel Electrophoresis ◽  
Affinity Capture ◽  
Bacterial Surface ◽  
Protective Antigens ◽  
Cell Enzyme

ABSTRACT The identification of the subset of outer membrane proteins exposed on the surface of a bacterial cell (the surfaceome) is critical to understanding the interactions of bacteria with their environments and greatly narrows the search for protective antigens of extracellular pathogens. The surfaceome of Leptospira was investigated by biotin labeling of viable leptospires, affinity capture of the biotinylated proteins, two-dimensional gel electrophoresis, and mass spectrometry (MS). The leptospiral surfaceome was found to be predominantly made up of a small number of already characterized proteins, being in order of relative abundance on the cell surface: LipL32 > LipL21 > LipL41. Of these proteins, only LipL32 had not been previously identified as surface exposed. LipL32 surface exposure was subsequently verified by three independent approaches: surface immunofluorescence, whole-cell enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Three other proteins, Q8F8Q0 (a putative transmembrane outer membrane protein) and two proteins of 20 kDa and 55 kDa that could not be identified by MS, one of which demonstrated a high degree of labeling potentially representing an additional, as-yet-uncharacterized, surface-exposed protein. Minor labeling of p31LipL45, GroEL, and FlaB1 was also observed. Expression of the surfaceome constituents remained unchanged under a range of conditions investigated, including temperature and the presence of serum or urine. Immunization of mice with affinity-captured surface components stimulated the production of antibodies that bound surface proteins from heterologous leptospiral strains. The surfaceomics approach is particularly amenable to protein expression profiling using small amounts of sample (<107 cells) offering the potential to analyze bacterial surface expression during infection.

scholarly journals Fosfomycin Reduces CD15s-Related Antigen Expression of Streptococcus pyogenes

1998 ◽  
Vol 42 (5) ◽  
pp. 1083-1087 ◽  
Author(s):  
Katsuhiko Hirota ◽  
Kinya Murakami ◽  
Ken Nemoto ◽  
Tsuneko Ono ◽  
Takashi Matsuo ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Surface Antigen ◽  
Phosphonic Acid ◽  
Laser Scanning ◽  
Antigen Expression ◽  
Host Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sialyl Lewis ◽  
Cell Enzyme ◽  
Selectin Family

ABSTRACT We have previously shown the immunological mimicry of human sialyl-Lewisx (CD15s) by a surface antigen ofStreptococcus pyogenes. This mimicking surface antigen may act as a ligand to the selectin family and may induce antibody production against CD15s on host cells, suggesting a possible role in the pathogenesis of S. pyogenes. In this study, the effects of antibiotics on the CD15s-related antigen expression of S. pyogenes were examined at a concentration below the MIC (sub-MIC). The amounts of CD15s on the surfaces of S. pyogenes cells and on the surfaces of S. pyogenesbiofilms were determined by a whole-cell enzyme-linked immunosorbent assay and by laser scanning fluorescence microscopy, respectively, by using an anti-CD15s monoclonal antibody. At the sub-MICs, fosfomycin (1R,2S-1,2-epoxypropyl phosphonic acid), its enantiomer (1S,2R-1,2-epoxypropyl phosphonic acid), and benzylpenicillin significantly inhibited the CD15s expression of all strains studied. The effects of fosfomycin and its enantiomer on biofilms were also observed by scanning electron microscopy. Incubation of S. pyogenes with the sub-MIC of fosfomycin or its enantiomer, which has no antibacterial activity, reduced the amount of CD15s on the biofilm surface and made it smooth. These results suggest that fosfomycin or its enantiomer might be useful for preventing S. pyogenes adherence to human CD15s receptors and the resulting immunological pathogenicity.

scholarly journals Colonization of the murine oropharynx by Streptococcus pyogenes is governed by the Rgg2/3 quorum sensing system

2020 ◽  
Author(s):  
Artemis Gogos ◽  
Michael J. Federle
Keyword(s):  
Quorum Sensing ◽  
Streptococcus Pyogenes ◽  
Vaccine Development ◽  
Wide Spectrum ◽  
Regulatory System ◽  
Transcriptional Activator ◽  
Upper Respiratory Tract ◽  
Sensing System ◽  
Molecular Therapeutics ◽  
Quorum Sensing System

AbstractStreptococcus pyogenes is a human-restricted pathogen most often found in the human nasopharynx. Multiple bacterial factors are known to contribute to persistent colonization of this niche, and many are important in mucosal immunity and vaccine development. In this work, mice were infected intranasally with transcriptional regulator mutants of the Rgg2/3 quorum sensing (QS) system—a peptide-based signaling system conserved in sequenced isolates of S. pyogenes. Deletion of the QS system’s transcriptional activator (Δrgg2) dramatically diminished the percentage of colonized mice while deletion of the transcriptional repressor (Δrgg3) increased the percentage of colonized mice compared to wild type. Stimulation of the QS system using synthetic pheromones prior to inoculation did not significantly increase the percentage of animals colonized, indicating that QS-dependent colonization is responsive to the intrinsic conditions within the host upper respiratory tract. Bacterial RNA extracted directly from oropharyngeal swabs and evaluated by quantitative RT-PCR subsequently confirmed QS upregulation within one hour of inoculation. In the nasal-associated lymphoid tissue (NALT), a muted inflammatory response to the Δrgg2 bacteria suggests that their rapid elimination failed to elicit the previously characterized response to intranasal inoculation of GAS. This work identifies a new transcriptional regulatory system governing the ability of S. pyogenes to colonize the nasopharynx and provides knowledge that could help lead to decolonization therapeutics.Author SummaryStreptococcus pyogenes is responsible for a wide spectrum of diseases ranging from common pharyngitis to infrequent invasive infections like necrotizing fasciitis. The ability of this microorganism to persist in the human oropharynx predisposes colonized individuals to a variety of superficial and invasive diseases which lead to significant morbidities and mortality. Identification of the regulatory systems that augment the bacteria’s ability to colonize the oropharynx provides potential targets against which molecular therapeutics can be designed. Here we show that the Rgg2/3 quorum sensing system, an interbacterial communication system, governs the ability of S. pyogenes to colonize the murine oropharynx. Disruption of the system’s transcriptional activator reduced colonization dramatically, eliminated the transcription of two sets of genes known to be activated by the Rgg2/3 system, and tempered the innate immune response seen when S. pyogenes is intranasally infected into the mouse.

scholarly journals Molecular Characteristics of Streptococcus pyogenes Isolated From Chinese Children With Different Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Dingle Yu ◽  
Yunmei Liang ◽  
Qinghua Lu ◽  
Qing Meng ◽  
Wenjian Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antibiotic Resistance ◽  
Streptococcus Pyogenes ◽  
Wide Spectrum ◽  
Expression Patterns ◽  
Virulence Gene ◽  
High Rate ◽  
Molecular Characteristics ◽  
Virulence Gene Expression ◽  
Obstructive Sleep

Streptococcus pyogenes is a bacterial pathogen that causes a wide spectrum of clinical diseases exclusively in humans. The distribution of emm type, antibiotic resistance and virulence gene expression for S. pyogenes varies temporally and geographically, resulting in distinct disease spectra. In this study, we analyzed antibiotic resistance and resistance gene expression patterns among S. pyogenes isolates from pediatric patients in China and investigated the relationship between virulence gene expression, emm type, and disease categories. Forty-two representative emm1.0 and emm12.0 strains (n = 20 and n = 22, respectively) isolated from patients with scarlet fever or obstructive sleep apnea-hypopnea syndrome were subjected to whole-genome sequencing and phylogenetic analysis. These strains were further analyzed for susceptibility to vancomycin. We found a high rate and degree of resistance to macrolides and tetracycline in these strains, which mainly expressed ermB and tetM. The disease category correlated with emm type but not superantigens. The distribution of vanuG and virulence genes were associated with emm type. Previously reported important prophages, such as φHKU16.vir, φHKU488.vir, Φ5005.1, Φ5005.2, and Φ5005.3 encoding streptococcal toxin, and integrative conjugative elements (ICEs) such as ICE-emm12 and ICE-HKU397 encoding macrolide and tetracycline resistance were found present amongst emm1 or emm12 clones from Shenzhen, China.

scholarly journals tRNA Modification by GidA/MnmE Is Necessary for Streptococcus pyogenes Virulence: a New Strategy To Make Live Attenuated Strains

2008 ◽  
Vol 76 (7) ◽  
pp. 3176-3186 ◽  
Author(s):  
Kyu Hong Cho ◽  
Michael G. Caparon
Keyword(s):  
Virulence Factors ◽  
Streptococcus Pyogenes ◽  
Vaccine Development ◽  
Translation Efficiency ◽  
Trna Modification ◽  
Necrosis Factor Alpha ◽  
New Strategy ◽  
In The Wild ◽  
Interleukin 23

ABSTRACT Studies directed at vaccine development and mucosal immunity against Streptococcus pyogenes would benefit from the availability of live attenuated strains. Our approach for production of candidate live attenuated strains was to identify mutations that did not alter growth in vitro and did not alter the overall complement of virulence factors produced but did result in reduced levels of expression of multiple secreted virulence factors. A global reduction but not elimination of expression would likely lead to attenuation while maximizing the number of antigenic targets available for stimulation of immunity. Adaptation of Tn5-based transposome mutagenesis to S. pyogenes with initial screening for reduced expression of the SpeB protease resulted in identification of mutations in gidA, which encodes an enzyme involved in tRNA modification. Reduced SpeB expression was due to delayed onset of speB transcription resulting from reduced translation efficiency of the message for RopB, a transcriptional activator. Overall, GidA− mutants had a nearly normal global transcription profile but expressed significantly reduced levels of multiple virulence factors due to impaired translation efficiencies. A translation defect was supported by the observation that mutants lacking MnmE, which functions in the same tRNA modification pathway as GidA, phenocopied GidA deficiency. The mutants stimulated a cytokine response in cultured macrophages identical to that in the wild type, with the exception of reduced levels of tumor necrosis factor alpha and interleukin-23. Significantly, GidA− mutants were highly attenuated in the murine ulcer model of soft tissue infection. These characteristics suggest that GidA pathway tRNA modification mutants are attractive candidates for further evaluation as live attenuated strains.

Antibodies against Specific Proteins of and Immobilizing Activity against Three Strains of Borrelia burgdorferi Sensu Lato Can Be Found in Symptomatic but Not in Infected Asymptomatic Dogs

2000 ◽  
Vol 38 (7) ◽  
pp. 2611-2621 ◽  
Author(s):  
Joppe W. R. Hovius ◽  
K. Emil Hovius ◽  
Anneke Oei ◽  
Dirk J. Houwers ◽  
Alje P. van Dam
Keyword(s):  
Borrelia Burgdorferi ◽  
Acute Phase ◽  
Bactericidal Activity ◽  
Borrelia Burgdorferi Sensu Lato ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blots ◽  
Cell Enzyme ◽  
Specific Proteins ◽  
Different Strains

In an area where Lyme disease is endemic in The Netherlands all dogs had positive titers by whole-cell enzyme-linked immunosorbent assay and appeared to be naturally infected by Borrelia burgdorferi sensu lato. To compare the antibody responses of symptomatic dogs and asymptomatic controls, we performed Western blots and in vitro immobilization assays to study antibody-dependent bactericidal activity. Strains from three different genospecies were employed as the antigen source: B. burgdorferi strain B31,Borrelia garinii strain A87S, and Borrelia afzelii strain pKo. Antibodies against flagellin (p41) and p39 for three strains were found in sera from both symptomatic and asymptomatic dogs and were therefore considered to be markers of exposure. Antibodies against p56 and p30 of strain B31, against p75, p58, p50, OspC, and p<19 of strain A87S, and against p56, p54, p45, OspB, p31, p26, and p<19 of strain pKo were found significantly more frequently in sera from symptomatic dogs younger than 8 years when the first symptoms were observed than in those from age-matched controls (P < 0.01). These antibodies were not found in preclinical sera and appeared during development of disease. Antibodies against OspA of strains B31 and A87S were only seen in acute-phase and convalescent sera from three dogs that recovered from disease. Incubation with 25% normal canine serum did not result in the immobilization of strains B31 and pKo, but partial immobilization of strain A87S (61% ± 24% [standard deviation] at 5 h) occurred. Seven of 15 sera from symptomatic dogs but none of the sera from 11 asymptomatic dogs had antibody-dependent immobilizing activity against one of the strains. Consecutive sera from one of these dogs immobilized two different strains. Antibody-mediated bactericidal serum was not seen before onset of disease, was strongest in the acute phase of disease, and fluctuated during chronic disease. From seven out of eight symptomatic dogs Borrelia DNA was amplified by PCR; in three of them the bactericidal activity was directed against one of the genospecies amplified from that dog; however, four PCR-positive dogs lacked bactericidal activity. In conclusion, dogs with symptomatic canine borreliosis have more-extensive antibody reactivity against Borrelia, as shown by both Western blotting and immobilization assays.

Urinary Claudin-1 Level as a Marker of Podocyte Injury in Patients with Proteinuria

QJM ◽  
2020 ◽  
Vol 113 (Supplement_1) ◽  
Author(s):  
M E Nasser ◽  
S M Shawky ◽  
E A M Sanad
Keyword(s):  
Wide Spectrum ◽  
Cell Junction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Podocyte Injury ◽  
Group Iii ◽  
Group I ◽  
Significant Difference ◽  
Diaphragm Cell ◽  
Control Study

Abstract Background The biology of claudins is a rapidly evolving field, and many intriguing questions remain unanswered. Although it had been assumed that the reason there are ≥24 isoforms of claudin is that each one has distinct permeability properties. The nephron displays a wide spectrum of claudins, whose distribution varies in each tubular segment. In diabetic nephropathy and glomerulonephritis the gene expression of claudin-1, is markedly upregulated in the podocyte, accompanied by a tighter filtration slit diaphragm (cell-cell junction made by the glomerular podocytes) and the appearance of TJ-like structures between the foot processes causing further podocytes injury and proteinuria. Aim of the work to assess urinary claudin -1 level as a marker of podocyte injury in patients with proteinuria. Patients and Methods it is a case control study which was conducted upon 90 subjects who were divided into three groups: group I included 30 patients with type II DM, group II included 30 patients with glomerulonephritis and group III had 30 healthy subjects as controls. Urinary claudin-1 level was measured by Enzyme linked Immunosorbent Assay (ELISA) Results In this study, we found that urinary claudin-1 level was significantly higher in diabetics group and GN group than in control group. In comparison between GN group and diabetic group, we found that urinary claudin-1 level was higher in GN group than in diabetics group but with no statistically significant difference between the two groups. Conclusion urinary claudin-1 level was significantly higher in diabetics and GN group and has positive correlation with uACR. So it can be used as marker of podocytes injury and proteinuria

scholarly journals Extracellular MIF, but not its homologue D-DT, promotes fibroblast motility independent of its receptor CD74/CD44

2020 ◽  
pp. jcs.217356
Author(s):  
Paweł Szczęśniak ◽  
Tamara Henke ◽  
Suada Fröhlich ◽  
Uwe Plessmann ◽  
Henning Urlaub ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Biological Activities ◽  
Wide Spectrum ◽  
Oxidoreductase Activity ◽  
Dopachrome Tautomerase ◽  
Associated Proteins ◽  
Signalling Transduction ◽  
Thiol Protein

Macrophage migration inhibitory factor (MIF) and its homologue D-dopachrome tautomerase (D-DT) are ubiquitous, pro-inflammatory cytokines with chemokine-like functions that coordinate a wide spectrum of biological activities like migration. Here, we biotin-tagged intracellular MIF/D-DT in vivo to identify important cytosolic interactors and found a plethora of actin cytoskeleton-associated proteins. While the CD74/CD44 receptor complex is essential for signalling transduction in fibroblasts by extracellular MIF/D-DT, our interactome data rather suggested direct effects. We thus investigated whether MIF/D-DT can modulate cell migration independent of CD74/CD44. To differentiate between receptor- and non-receptor-mediated motility, we treated fibroblasts that are deficient in CD74 and CD44 or that express both proteins with recombinant MIF/D-DT. Interestingly, only MIF could stimulate chemokinesis in the presence or absence of CD74/CD44. The pro-migratory effects of MIF depended on lipid raft/caveolae-mediated but not clathrin-mediated endocytosis, on its tautomerase activity and, likely, on its thiol protein oxidoreductase activity. As MIF treatment restrained actin polymerisation in vitro our findings establish a new intracellular role for MIF/D-DT in driving cell motility by modulating the actin cytoskeleton.

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