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 Identification of 19 Polymorphic Major Outer Membrane Protein Genes and Their Immunogenic Peptides in Ehrlichia ewingii for Use in a Serodiagnostic Assay

2007 ◽  
Vol 15 (3) ◽  
pp. 402-411 ◽  
Author(s):  
Chunbin Zhang ◽  
Qingming Xiong ◽  
Takane Kikuchi ◽  
Yasuko Rikihisa
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Genomic Region ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serologic Test ◽  
Bacterial Surface ◽  
Immunogenic Peptides ◽  
Genes Encoding ◽  
Molecular Masses ◽  
Touchdown Pcr

ABSTRACT Ehrlichia ewingii, a tick-transmitted rickettsia previously known only as a canine pathogen, was recently recognized as a human pathogen. E. ewingii has yet to be cultivated, and there is no serologic test available to diagnose E. ewingii infection. Previously, a fragment (505 bp) of a single E. ewingii gene homologous to 1 of 22 genes encoding Ehrlichia chaffeensis immunodominant major outer membrane proteins 1 (OMP-1s)/P28s was identified. The purposes of the present study were to (i) determine the E. ewingii omp-1 gene family, (ii) determine each OMP-1-specific peptide, and (iii) analyze all OMP-1 synthesized peptides for antigenicity. Using nested touchdown PCR and a primer walking strategy, we found 19 omp-1 paralogs in E. ewingii. These genes are arranged in tandem downstream of tr1 and upstream of secA in a 24-kb genomic region. Predicted molecular masses of the 19 mature E. ewingii OMP-1s range from 25.1 to 31.3 kDa, with isoelectric points of 5.03 to 9.80. Based on comparative sequence analyses among OMP-1s from E. ewingii and three other Ehrlichia spp., each E. ewingii OMP-1 oligopeptide that was predicted to be antigenic, bacterial surface exposed, unique in comparison to the other E. ewingii OMP-1s, and distinct from those of other Ehrlichia spp. was synthesized for use in an enzyme-linked immunosorbent assay. Plasmas from experimentally E. ewingii-infected dogs reacted significantly with most of the OMP-1-specific peptides, indicating that multiple OMP-1s were expressed and immunogenic in infected dogs. The results support the utility of the tailored OMP-1 peptides as E. ewingii serologic test antigens.

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 Differential Expression and Sequence Conservation of the Anaplasma marginale msp2 Gene Superfamily Outer Membrane Proteins

2006 ◽  
Vol 74 (6) ◽  
pp. 3471-3479 ◽  
Author(s):  
Susan M. Noh ◽  
Kelly A. Brayton ◽  
Donald P. Knowles ◽  
Joseph T. Agnes ◽  
Michael J. Dark ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Differential Expression ◽  
Outer Membrane Proteins ◽  
Anaplasma Marginale ◽  
Surface Proteins ◽  
Transcriptional Analysis ◽  
Mammalian Host ◽  
Anaplasma Ovis ◽  
Sequence Comparisons

ABSTRACT Bacterial pathogens in the genera Anaplasma and Ehrlichia encode a protein superfamily, pfam01617, which includes the predominant outer membrane proteins (OMPs) of each species, major surface protein 2 (MSP2) and MSP3 of Anaplasma marginale and Anaplasma ovis, Anaplasma phagocytophilum MSP2 (p44), Ehrlichia chaffeensis p28-OMP, Ehrlichia canis p30, and Ehrlichia ruminantium MAP1, and has been shown to be involved in both antigenic variation within the mammalian host and differential expression between the mammalian and arthropod hosts. Recently, complete sequencing of the A. marginale genome has identified an expanded set of genes, designated omp1-14, encoding new members of this superfamily. Transcriptional analysis indicated that, with the exception of the three smallest open reading frames, omp2, omp3, and omp6, these superfamily genes are transcribed in A. marginale-infected erythrocytes, tick midgut and salivary glands, and the IDE8 tick cell line. OMPs 1, 4, 7 to 9, and 11 were confirmed to be expressed as proteins by A. marginale within infected erythrocytes, with expression being either markedly lower (OMPs 1, 4, and 7 to 9) or absent (OMP11) in infected tick cells, which reflected regulation at the transcript level. Although the pfam01617 superfamily includes the antigenically variable MSP2 and MSP3 surface proteins, analysis of the omp1-14 sequences throughout a cycle of acute and persistent infection in the mammalian host and tick transmission reveals a high degree of conservation, an observation supported by sequence comparisons between the St. Maries strain and Florida strain genomes.

scholarly journals A Proline-Rich Region with a Highly Periodic Sequence in Streptococcal β Protein Adopts the Polyproline II Structure and Is Exposed on the Bacterial Surface

2002 ◽  
Vol 184 (22) ◽  
pp. 6376-6383 ◽  
Author(s):  
Thomas Areschoug ◽  
Sara Linse ◽  
Margaretha Stålhammar-Carlemalm ◽  
Lars-Olof Hedén ◽  
Gunnar Lindahl
Keyword(s):  
Cell Wall ◽  
Surface Expression ◽  
Surface Proteins ◽  
Rich Region ◽  
Bacterial Surface ◽  
Polyproline Ii ◽  
Β Protein ◽  
Typical Sequence ◽  
Sequence Periodicity ◽  
Proline Rich Region

ABSTRACT Proline-rich regions have been identified in many surface proteins of pathogenic streptococci and staphylococci. These regions have been suggested to be located in cell wall-spanning domains and/or to be required for surface expression of the protein. Because little is known about these regions, which are found in extensively studied and biologically important surface proteins, we characterized the proline-rich region in one such protein, the β protein of group B streptococci. The proline-rich region in β, designated the XPZ region, has a proline at every third position, and the sequence is highly periodic in other respects. Immunochemical analysis showed that the XPZ region was not associated with the cell wall but was exposed on the bacterial surface. Moreover, characterization of a β mutant lacking the XPZ region demonstrated that this region was not required for surface expression of the β protein. Comparison of the XPZ region in different β proteins showed that it varied in size but always retained the typical sequence periodicity. Circular dichroism spectroscopy indicated that the XPZ region had the structure of a polyproline II helix, an extended and solvent-exposed structure with exactly three residues per turn. Because of the three-residue sequence periodicity in the XPZ region, it is expected to be amphipathic and to have distinct nonpolar and polar surfaces. This study identified a proline-rich structure with unique properties that is exposed on the surface of an important human pathogen.

The search forBrachyspiraouter membrane proteins that interact with the host

2001 ◽  
Vol 2 (1) ◽  
pp. 19-30 ◽  
Author(s):  
Darren J. Trott ◽  
David P. Alt ◽  
Richard L. Zuerner ◽  
Michael J. Wannemuehler ◽  
Thaddeus B. Stanton
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Membrane Vesicle ◽  
Outer Membrane Proteins ◽  
Indirect Evidence ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Surface Proteins ◽  
Brachyspira Pilosicoli ◽  
Variable Surface

AbstractLittle is known about the outer membrane structure ofBrachyspira hyodysenteriae and Brachyspira pilosicolior the role of outer membrane proteins (OMPs) in host colonization and the development of disease. The isolation of outer membrane vesicles fromB. hyodysenteriaehas confirmed that cholesterol is a significant outer membrane constituent and that it may impart unique characteristics to the lipid bilayer structure, including a reduced density. Unique proteins that have been identified in theB. hyodysenteriaeouter membrane include the variable surface proteins (Vsp) and lipoproteins such as SmpA and BmpB. While the function of these proteins remains to be determined, there is indirect evidence to suggest that they may be involved in immune evasion. These data may explain the ability of the organism to initiate chronic infection. OMPs may be responsible for the unique attachment ofB. pilosicolito colonic epithelial cells; however, the onlyB. pilosicoliOMPs that have been identified to date are involved in metabolism. In order to identify furtherB. pilosicoliOMPs we have isolated membrane vesicle fractions from porcine strain 95–1000 by osmotic lysis and isopycnic centrifugation. The fractions were free of contamination by cytoplasm and fla-gella and contained outer membrane. Inner membrane contamination was minimal but could not be completely excluded. An abundant 45-kDa, heat-modifiable protein was shown to have significant homology withB. hyodysenteriaeVsp, and monoclonal antibodies were produced that reacted with fiveB. pilosicoli-specificmembrane protein epitopes. The first of these proteins to be characterized is a unique surface-exposed lipoprotein.

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