Characterization of monoclonal antibodies to Yersinia enterocolitica iron-regulated proteins

1995 ◽  
Vol 41 (7) ◽  
pp. 562-571 ◽  
Author(s):  
Cora Kooi ◽  
Pamela A. Sokol
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Yersinia Enterocolitica ◽  
Iron Acquisition ◽  
Proteinase K ◽  
Infection Model ◽  
Mouse Infection Model ◽  
Molecular Masses ◽  
Proteinase K Treatment

Yersinia enterocolitica 1165 (0:8) expressed several iron-regulated proteins with molecular masses of 240, 194, 80, 79, 70, and 67 kDa. These proteins were not detected in cells grown in iron-rich conditions. Cell surface iodination indicated that the 240- and 190-kDa proteins (HMWPs) were not surface exposed, whereas the 67- and 70-kDa proteins appeared to be exposed to the cell surface. Incubation with iron protected the 67- and 70-kDa proteins from proteinase K treatment, suggesting that they may be involved in iron acquisition. Monoclonal antibodies (MAbs) were produced against the HMWPs and the 67-kDa iron-regulated protein. MAbs to the HMWPs not only recognized the 240- and 194-kDa proteins but also reacted with the 67- and 70-kDa iron-regulated proteins. Similarly, MAbs to the 67-kDa protein reacted with the 67- and 70-kDa proteins and the HMWPs, suggesting that these iron-regulated proteins are related immunologically. In addition, the MAbs recognized the 67- and 70-kDa proteins and HMWPs from other Y. enterocolitica serotypes, suggesting that the antigenic sites recognized on these iron-regulated proteins are conserved. The MAbs examined did not inhibit iron binding or iron uptake and did not provide protection against a Y. enterocolitica 1165 (0:8) infection in a systemic mouse infection model. Although these MAbs were not protective in this model, these iron-regulated proteins may play a role in iron acquisition and virulence, but the MAbs examined are probably not directed against epitopes involved in iron acquisition or virulence.Key words: Yersinia enterocolitica, monoclonal antibodies, iron-regulated proteins.

scholarly journals Direct Involvement of Type II Secretion System in Extracellular Translocation of Shewanella oneidensis Outer Membrane Cytochromes MtrC and OmcA

2008 ◽  
Vol 190 (15) ◽  
pp. 5512-5516 ◽  
Author(s):  
Liang Shi ◽  
Shuang Deng ◽  
Matthew J. Marshall ◽  
Zheming Wang ◽  
David W. Kennedy ◽  
...  
Keyword(s):  
Cell Surface ◽  
Shewanella Oneidensis ◽  
Secretion System ◽  
Proteinase K ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Direct Involvement ◽  
Type Ii Secretion System ◽  
Extracellular Release ◽  
Proteinase K Treatment

ABSTRACT MtrC and OmcA are cell surface-exposed lipoproteins important for reducing solid metal oxides. Deletions of type II secretion system (T2SS) genes reduced their extracellular release and their accessibility to the proteinase K treatment, demonstrating the direct involvement of T2SS in translocation of MtrC and OmcA to the bacterial cell surface.

scholarly journals Identification and Characterization of Di- and Tripeptide Transporter DtpT of Listeria monocytogenes EGD-e

2005 ◽  
Vol 71 (10) ◽  
pp. 5771-5778 ◽  
Author(s):  
Jeroen A. Wouters ◽  
Torsten Hain ◽  
Ajub Darji ◽  
Eric Hüfner ◽  
Henrike Wemekamp-Kamphuis ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Opportunistic Infections ◽  
Essential Amino Acids ◽  
Defined Medium ◽  
Infection Model ◽  
Intracellular Pathogen ◽  
Mouse Infection Model ◽  
Stress Protection ◽  
Identification And Characterization

ABSTRACT Listeria monocytogenes is a gram-positive intracellular pathogen responsible for opportunistic infections in humans and animals. Here we identified and characterized the dtpT gene (lmo0555) of L. monocytogenes EGD-e, encoding the di- and tripeptide transporter, and assessed its role in growth under various environmental conditions as well as in the virulence of L. monocytogenes. Uptake of the dipeptide Pro-[14C]Ala was mediated by the DtpT transporter and was abrogated in a ΔdtpT isogenic deletion mutant. The DtpT transporter was shown to be required for growth when the essential amino acids leucine and valine were supplied as peptides. The protective effect of glycine- and proline-containing peptides during growth in defined medium containing 3% NaCl was noted only in L. monocytogenes EGD-e, not in the ΔdtpT mutant strain, indicating that the DtpT transporter is involved in salt stress protection. Infection studies showed that DtpT contributes to pathogenesis in a mouse infection model but has no role in bacterial growth following infection of J774 macrophages. These studies reveal that DptT may contribute to the virulence of L. monocytogenes.

scholarly journals Production and Characterization of Two Monoclonal Antibodies Specific for Plasmopara halstedii

2000 ◽  
Vol 66 (8) ◽  
pp. 3277-3282 ◽  
Author(s):  
S. Bouterige ◽  
R. Robert ◽  
J. P. Bouchara ◽  
A. Marot-Leblond ◽  
V. Molinero ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Detection System ◽  
Sandwich Elisa ◽  
Plasmopara Viticola ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasmopara Halstedii ◽  
Race 1 ◽  
Molecular Masses ◽  
Fungal Antigens

ABSTRACT Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races ofP. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.

Characterization of Monoclonal Antibodies Against Cell Surface Molecules Associated with Cytotoxic Activity of Natural and Activated Killer Cells and Cloned CTL Lines

Hybridoma ◽  
1983 ◽  
Vol 2 (4) ◽  
pp. 423-437 ◽  
Author(s):  
HERGEN SPITS ◽  
GERRIT KEIZER ◽  
JANNIE BORST ◽  
COX TERHORST ◽  
ANNEMARIE HEKMAN ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Cytotoxic Activity ◽  
Cell Surface Molecules ◽  
Killer Cells ◽  
Surface Molecules

scholarly journals Patterns of gene expression in schistosomes: localization by whole mountin situhybridization

Parasitology ◽  
2007 ◽  
Vol 134 (11) ◽  
pp. 1589-1597 ◽  
Author(s):  
G. P. DILLON ◽  
J. C. ILLES ◽  
H. V. ISAACS ◽  
R. A. WILSON
Keyword(s):  
Gene Expression ◽  
Cathepsin L ◽  
Expression Patterns ◽  
Proteinase K ◽  
Fluorescent Substrate ◽  
Rna Probes ◽  
Fast Red ◽  
Proteinase K Treatment

SUMMARYAs a consequence of comprehensive transcriptome analysis followed by sequencing and draft assembly of the genome, the emphasis of schistosome research is shifting from the identification of genes to the characterization of their functions and interactions. Developmental biologists have long used whole mountin situhybridization (WISH) to determine gene expression patterns, as a vital tool for formulating and testing hypotheses about function. This paper describes the application of WISH to the study of gene expression in larval and adult schistosomes. Fixed worms were permeablized by proteinase K treatment for hybridization with digoxygenin-labelled RNA probes, with binding being detected by alkaline phosphatase-coupled anti-digoxygenin antibodies, and BM Purple substrate. Discrete staining patterns for the transcripts of the molecules Sm29, cathepsin L, antigen 10.3 and chorion were observed in the tegument cell bodies, gut epithelium, oesophageal gland and vitelline lobules, respectively, of adult worms. Transcripts of the molecules SGTP4, GP18-22 and cathepsin L were localized to tegument cell bodies and embryonic gut, respectively, of lung schistosomula. We also showed that Fast Red TR fluorescent substrate can refine the pattern of localization permitting use of confocal microscopy. We believe that method of WISH will find broad application, in synergy with other emerging post-genomic techniques, such as RNA interference, to studies focused at increasing our molecular understanding of schistosomes.

Characterization of a virulence-associated and cell-wall-located DNase of Streptococcus pyogenes

Microbiology ◽  
2010 ◽  
Vol 156 (1) ◽  
pp. 184-190 ◽  
Author(s):  
Tadao Hasegawa ◽  
Masaaki Minami ◽  
Akira Okamoto ◽  
Ichiro Tatsuno ◽  
Masanori Isaka ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Pyogenes ◽  
Protein A ◽  
Insoluble Fraction ◽  
Infection Model ◽  
Two Dimensional Gel Electrophoresis ◽  
Mouse Infection Model ◽  
Peptide Mass ◽  
A Cell

We investigated culture supernatant proteins from the M1 serotype of Streptococcus pyogenes by two-dimensional gel electrophoresis and peptide mass mapping analysis, and characterized the single protein spots. Among them, we analysed the Spy0747 protein. This protein is homologous to the SsnA protein, a cell-wall-located DNase expressed in Streptococcus suis serotype 2. We designated the Spy0747 protein as SpnA. SpnA protein was also detected in the insoluble fraction of whole-cell lysates using shotgun proteomic analysis, suggesting that SpnA is also located in the cell wall. SpnA was expressed as a glutathione S-transferase-fusion protein in Escherichia coli. We confirmed that the recombinant protein had DNase activity that was dependent on Ca2+ and Mg2+, like SsnA. Blood bactericidal assays and mouse infection model experiments showed that the spnA knockout strain was less virulent than the parental strain, thus suggesting that SpnA could play an important role in virulence. Using PCR, we found that the spnA gene was present in all clinical S. pyogenes strains we examined. Our results, together with a previous report identifying Spy0747 as a surface-associated protein, suggest that SpnA is an important cell-wall-located DNase that is generally produced in S. pyogenes and is involved in virulence.

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