scholarly journals Comparative studies of Yersinia pestis outer membrane isolation techniques and their potential use in plague epidemiology

1990 ◽  
Vol 32 (2) ◽  
pp. 78-83 ◽  
Author(s):  
Frederico Guilherme Coutinho Abath ◽  
Luís Carlos de Sousa Ferreira
Keyword(s):  
Yersinia Pestis ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Patterns ◽  
Isolation Techniques ◽  
Sds Page ◽  
Triton X ◽  
Potential Use

In the present study three techniques for obtaining outer membrane enriched fractions from Yersinia pestis were evaluated. The techniques analysed were: differential solubilization of the cytoplasmic membrane with Sarkosyl or Triton X-100, and centrifugation in sucrose density gradients. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane isolated by the different methods resulted in similar protein patterns. The measurement of NADH-dehydrogenase and succinate dehydrogenase (inner membrane enzymes) indicated that the outer membrane preparations obtained by the three methods were pure enough for analytical studies. In addition, preliminary evidences on the potential use of outer membrane proteins for the identification of geographic variants of Y. pestis wild isolates are presented.

scholarly journals Establishing a Direct Role for the Bartonella bacilliformis Invasion-Associated Locus B (IalB) Protein in Human Erythrocyte Parasitism

2001 ◽  
Vol 69 (7) ◽  
pp. 4373-4381 ◽  
Author(s):  
Sherry A. Coleman ◽  
Michael F. Minnick
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Protein Localization ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Amino Acid Sequence Similarity ◽  
Bartonella Bacilliformis ◽  
Sds Page

ABSTRACT The invasion-associated locus A and B genes (ialAB) ofBartonella bacilliformis were previously shown to confer an erythrocyte-invasive phenotype upon Escherichia coli, indirectly implicating their role in virulence. We report the first direct demonstration of a role for ialB as a virulence factor in B. bacilliformis. The presence of a secretory signal sequence and amino acid sequence similarity to two known outer membrane proteins involved in virulence suggested that IalB was an outer membrane protein. To develop an antiserum for protein localization, the ialB gene was cloned in frame into an expression vector with a six-histidine tag and under control of thelacZ promoter. The IalB fusion protein was purified by nickel affinity chromatography and used to raise polyclonal antibodies. IalB was initially localized to the bacterial membrane fraction. To further localize IalB, B. bacilliformis inner and outer membranes were fractionated by sucrose density gradient centrifugation and identified by appearance, buoyant density (ρ), and cytochromeb content. Inner and outer membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and IalB was positively identified by Western blot. Contrary to expectations, IalB was localized to the inner membrane of the pathogen. To directly demonstrate a role for IalB in erythrocyte parasitism, the B. bacilliformis ialB gene was disrupted by insertional mutagenesis. The resulting ialB mutant strain was complemented in trans with a replicative plasmid encoding the full-length ialB gene. PCR and high-stringency DNA hybridization confirmed mutagenesis and transcomplementation events. Abrogation and restoration of ialB expression was verified by SDS-PAGE and immunoblotting. In vitro virulence assays showed that mutagenesis of ialB decreased bacterial association and invasion of human erythrocytes by 47 to 53% relative to controls. Transcomplementation of ialB restored erythrocyte association and invasion rates to levels observed in the parental strain. These data provide direct evidence for IalB's role in erythrocyte parasitism and represent the first demonstration of molecular Koch's postulates for a Bartonella species.

Variability of Low-Molecular-Weight, Heat-Modifiable Outer Membrane Proteins of Neisseria meningitidis

1980 ◽  
Vol 30 (3) ◽  
pp. 642-648
Author(s):  
J. T. Poolman ◽  
S. De Marie ◽  
H. C. Zanen
Keyword(s):  
Molecular Weight ◽  
Outer Membrane ◽  
High Molecular Weight ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Low Molecular Weight ◽  
Molecular Weights ◽  
Sds Page ◽  
Index Cases

Analysis of major outer membrane protein (MOMP) profiles of various meningococci by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of 0 to 2 low-molecular-weight, heat-modifiable MOMPs (molecular weight, 25,000 to 32,000) and 1 to 3 high-molecular-weight MOMPs (molecular weight, 32,000 to 46,000). Heat modifiability was investigated by comparing MOMP profiles after heating in SDS solutions at 100°C for 5 min or at 40°C for 1 h. Low-molecular-weight MOMPs shifted to higher apparent molecular weights after being heated at 100°C. Heat modifiability of high-molecular-weight MOMPs varied among strains; whenever modified these proteins shifted to lower apparent molecular weights after complete denaturation. Variability of low-molecular-weight, heat-modifiable MOMPs was demonstrated when MOMP profiles were compared of (i) isolates from index cases and associated cases and carriers among contacts, (ii) different isolates from the same individual, and (iii) isolates from a small epidemic caused by serogroup W-135. In some cases high-molecular-weight MOMPs revealed quantitative differences among related strains. The observed variability and quantitative differences indicate that MOMP serotyping and typing on the basis of SDS-PAGE profiles (PAGE typing) need careful reevaluation.

scholarly journals Comparative Proteomics Study of Outer Membrane Proteins from three Pathogenic Leptospira Species used in Vaccine

2015 ◽  
Vol 5 (3) ◽  
pp. 753-760
Author(s):  
Khosrow Aghaiypour Kolyani ◽  
Rahman Shokri
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Double Diffusion ◽  
Pathogenic Leptospira ◽  
Sds Page ◽  
Antigenic Properties ◽  
Molecular Masses

 Background and Objective: Outer Membrane Proteins (OMPs) have an important role in pathogenecity and immunogenecity of Leptospira introgans. The aim of this study was chemical and immunological analysis of OMP extracted from three vaccinal strains of pathogenic Leptospira (L. canicola, L. grippotyphosa & L. sejroae hardjo) by electrophoresis and western blotting.Materials and Methods: Outer membrane enriched fractions that are insoluble in sodium N- lauryl sarcosinate isolated and studied by Sodium dodecyl sulfate – polyacrylamide gel electrophoresis. For identifying of antigenic properties of different proteins in three strains, antiserums developed in rabbit against the whole three valent vaccine and also  specific OMPs rom each of three Leptospira strains. The antiserum was used for immunological studies with immuno double diffusion and western blotting.Results: in SDS-PAGE five common protein bands with approximate molecular masses of 75,36,25,23 and 19 KDa were identified. After western blot studies identified four or five immunogenic band. In general, antiserum against L. grippotyphosa OMP has the highest ability in detecting common bands between three strains.Conclusion: Chemical and immunological comparisons between OMPs of the three strains which are being used in the commercial vaccine, provided useful documents to assess and maybe improve vaccine quality.  It indicated that OMPs are one of the major Leptospira component contributing in immunity and protectivity. Comparison between individual OMPs of each serotype revealed that L. grippotyphosa ones could contribute more in vaccine induced protectively in comparison with the other two serotypes.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

Protein Expression in Vibrio parahaemolyticus 690 Subjected to Sublethal Heat and Ethanol Shock Treatments

2008 ◽  
Vol 71 (11) ◽  
pp. 2289-2294 ◽  
Author(s):  
MING-LUN CHIANG ◽  
WEI-LI HO ◽  
ROCH-CHUI YU ◽  
CHENG-CHUN CHOU
Keyword(s):  
Heat Shock ◽  
Vibrio Parahaemolyticus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Test Organism ◽  
Protein Patterns ◽  
One Dimensional ◽  
Sds Page ◽  
Molecular Masses ◽  
Stressed Cells

Cells of Vibrio parahaemolyticus 690 were subjected either to heat shock at 42°C for 45 min or to ethanol shock in the presence of 5% ethanol for 60 min. The protein profiles of the unstressed and stressed V. parahaemolyticus cells were compared. Additionally, the induction of DnaK- and GroEL-like proteins in the unstressed and stressed cells of V. parahaemolyticus was also examined. Analysis with one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) indicated that three proteins with molecular masses of 93, 77, and 58 kDa were induced by both heat shock and ethanol shock. The protein patterns revealed by two-dimensional electrophoresis were more detailed than those revealed by one-dimensional SDS-PAGE. It was found that heat shock and ethanol shock affected the expression of a total of 28 proteins. Among them, four proteins with molecular masses of 94, 32.1, 26.7, and 25.7 kDa were enhanced by both heat shock and ethanol shock. Furthermore, immunoblot analysis showed the presence of a GroEL-like protein with a molecular mass of 61 kDa in the test organism, with the heat-shocked and ethanol-shocked cells producing a GroEL-like protein in a larger quantity than the unstressed cells. However, DnaK-like protein was not detectable in either the unstressed or the stressed cells.

scholarly journals Identification of Polymorphic Outer Membrane Proteins of Chlamydia psittaci 6BC

2001 ◽  
Vol 69 (4) ◽  
pp. 2428-2434 ◽  
Author(s):  
Regina J. Tanzer ◽  
David Longbottom ◽  
Thomas P. Hatch
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Disulfide Bonds ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Late Phase ◽  
Sodium Dodecyl ◽  
Chlamydia Psittaci ◽  
Developmental Cycle ◽  
Lauryl Sarcosine

ABSTRACT The genomes of Chlamydia spp. encode a family of putative outer membrane proteins, referred to as polymorphic outer membrane proteins (POMPs), which may play a role in the avoidance of host immune defenses. We analyzed avian strain 6BC of Chlamydia psittaci by polyacrylamide gel electrophoresis for the expression of POMPs. At least six putative POMPs were identified on the basis of their size (90 to 110 kDa) and labeling with an outer membrane-specific probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Three of the putative POMPs reacted with antiserum raised against a recombinant ovine C. psittaci strain POMP, and two possessed surface-exposed, trypsin-sensitive sites. The POMPs were dependent on disulfide bonds for their maintenance in sodium lauryl sarcosine- and sodium dodecyl sulfate-insoluble complexes but did not appear to be interpeptide disulfide bond cross-linked. The putative POMPs were found to be synthesized during the late phase of the chlamydial developmental cycle, cotemporally with the cysteine-rich doublet periplasmic proteins.

Outer membrane proteins from Serratia marcescens

1993 ◽  
Vol 39 (1) ◽  
pp. 108-111 ◽  
Author(s):  
Marta Puig ◽  
Carme Fusté ◽  
Miquel Viñas
Keyword(s):  
Membrane Proteins ◽  
Serratia Marcescens ◽  
Outer Membrane ◽  
Nutrient Availability ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cultural Conditions

The outer membrane proteins (OMPs) of several strains of Serratia marcescens have been studied by sodium dodecyl sulphate – urea – polyacrylamide gel electrophoresis. Four major OMPs, named Omp1, Omp2, Omp3, and OmpA (42, 40, 39, and 37 kDa, respectively), have been visualized. The relative proportions of Omp2 and Omp3 depend on cultural conditions (temperature of incubation, osmolarity, and nutrient availability).Key words: Serratia marcescens, outer membrane proteins, porin.

scholarly journals MPB70 and MPB83 as Indicators of Protein Localization in Mycobacterial Cells

1998 ◽  
Vol 66 (1) ◽  
pp. 289-296 ◽  
Author(s):  
Morten Harboe ◽  
Harald G. Wiker ◽  
Gunni Ulvund ◽  
Bent Lund-Pedersen ◽  
Åse Bengård Andersen ◽  
...  
Keyword(s):  
Protein Localization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Culture Fluid ◽  
Secreted Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sds Page ◽  
Shock Proteins ◽  
Triton X

ABSTRACT Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates ofMycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

scholarly journals Evaluation of numerical analysis of SDS-PAGE of protein patterns for typingEnterobacter cloacae

1989 ◽  
Vol 103 (2) ◽  
pp. 265-274 ◽  
Author(s):  
M. Costas ◽  
L. L. Sloss ◽  
R. J. Owen ◽  
M. A. Gaston
Keyword(s):  
Numerical Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Phage Typing ◽  
Clinical Isolates ◽  
Protein Patterns ◽  
One Dimensional ◽  
Sds Page ◽  
Typing Methods ◽  
Type Strains

SUMMARYTwenty cultures comprising 13 clinical isolates ofEnterobacter cloacaefrom two hospitals. the type and another reference stain ofE. cloacaeand the type strains of four otherEnterobactersp. and ofEscherichia coli, were characterized by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) of whole-cell proteins. The protein patterns were highly reproducible and were used as the basis of a numerical analysis which divided the clinical isolates into nine clearly defined protein types. Comparison with established typing methods indicated that the discrimination of SDS-PAGE was similar to that achieved with conventional typing methods and all strain groups recognized by combined sero/phage typing were also found by SDS-PAGE. In addition, protein typing sub-divided a group of four serotype O3 isolates that were difficult to distinguish by phage typing. We conclude that high-resolution SDS-PAGE of proteins provides an effective method of typing isolates ofE. cloacae.

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