In silico prediction of exposure amino acid sequences of outer inflammatory protein A of Helicobacter pylori for surface display on Eschierchia coli.

BACKGROUND: Outer inflammatory protein A (OipA) is an outer membrane protein of Helicobacter pylori that is involved in inducing IL-8 and intracellular signaling. In this study, we have predicted exposure amino acid sequences of OipA for insertion in permissive sites of CstH subunit of Eschierchia coli CS3 pilli for bacterial surface display. MATERIALS AND METHODS: Databases: National Center for Biotechnology Institute and Protein Data Bank. Servers: PHD, SABLE, GOR 4, SignalP3.0, TBBpred, PRODIV-TMHMM, TMRPres2D, CPH Models, PHYRE, GETAREA, VADAR, Pep state and pep window. Software: Swiss PDB viewer and Discovery studio. RESULTS: In silico prediction of exposure amino acid sequences of OipA led to detection of six sequences of amino acid, 76-87, 106-112, 170-182, 222-230, 242-258, and 278-290. These sequences inserted between amino acid sequences 66-67, 100-101, and 109-110 of CstH that were predicted by Eskandari et al. as permissive sites of CstH. CONCLUSION: OipA has the ability to induce IL-8 from gastric epithelial cells and some papers are mentioned that this outer membrane protein involve to attachment and intracellular signaling. Receptor of OipA and adhesion motifs on this protein is unknown. Detection of exposure motifs aids to recognition of adhesion motifs and receptor of OipA on gastric epithelial cells. In this study, we have predicted exposure amino acid sequences for insert to subunit CstH of CS3 pilli E. coli for surface display.

Phenotypic & genotypic conservation of ompL1 & lipL41 among leptospiral isolates of Andaman Islands.

BACKGROUND & OBJECTIVES: The leptospiral antigens that are conserved among the diverse pathogenic leptospires have potential importance in the development of new serodiagnostic and immunoprotective strategies. The present study was therefore carried out to find out the phenotypic conservation of the leptospiral proteins OmpL1 and LipL41, and the genetic conservation of ompL1 and lipL41 genes among the leptospiral isolates of Andaman Islands and among the reference strains. METHODS: In one dimensional SDS-PAGE the leptospiral samples prepared from strains of various leptospiral serovars were run and transferred on to nitrocellulose paper and probed with pooled convalescent phase human sera to find out the phenotypic conservation of the protein fragments at 31 and 41 kDa. Further, the proteins were indirectly confirmed as OmpL1 and LipL41 by using specific rabbit hyperimmune sera. Specific primers were utilized to amplify the fragments to study the genetic conservation of ompL1 and lipL41. Further, these two fragments were sequenced and BLAST analysis was done with the whole genome of Leptospira interrogans serovar Lai for comparison. RESULTS: Analysis of individual immunoblots using patient sera showed that the OmpL1 and LipL41 were conserved among all the isolates used in the study. Further, these two proteins were probed with specific rabbit hyperimmune sera of OmpL1 and LipL41 for confirming the fragments and it was found to be conserved among all the isolates. The PCR based amplification further showed that the genes ompL1 and lipL41 were conserved among the leptospiral isolates studied. Sequencing followed by BLAST analysis of these showed 97 per cent similarity with the whole genome sequence and low score values in comparison with other bacterial species. INTERPRETATION & CONCLUSION: The antigenic and genetic conservation of the two proteins, OmpL1 and LipL41, indicated that these could be potential candidates for development of diagnostic test systems for leptospirosis.

Heat modifiability of outer membrane protein of Pasteurella multocida serotype B:2.

Outer membrane proteins (OMP) are generally porins, functioning as molecular sieves assisting in the transmembrane transportation. Heat modifiable characteristics of OMP from P. multocida B: 2 have been explored to know their basic characteristics on event of temperature rise. A major band of 32 kDa and two minor bands of approximately 39 and approximately 28 kDa were found to be heat modifiable. It is suggested that boiling at 100 degrees C in presence of beta mercaptoethanol for 5 min is sufficient for characterisation of OMP by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis.

Structural, functional and immunological studies on a polymeric bacterial protein

The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-A resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.

Amino acid sequences of proteins from Leptospira serovar pomona

This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.

Caracterización de un brote de Shigella boydii 14 por primera vez en Cuba / Characterization of an aoutbreak of Shigella boydii 14 for the first time in Cuba

Se propuso la caracterización de 14 cepas de Shigella boydii 14 aisladas de pacientes con enfermedad diarreica aguda mediante sus plásmidos de resistencia y de las proteínas de la membrana externa presentes en ellas. Se realizó la determinación de la susceptibilidad antimicrobiana por el método de concentración mínima inhibitoria, la extracción de plásmidos R fue según Manaitis, los extractos proteicos de las cepas se obtuvieron según el método de Blaser modificado y las proteínas de la membrana externa fueron separadas por SDS-PAGE por el método de Laemmli. Se comprobó que las cepas resultaron resistentes a la ampicilina (100 porciento), la tetraciclina (70 porciento) y al cotrimoxazol (50 porciento), y sensibles al ácido nalidíxico y a la ciprofloxacina. Se observó la presencia de plásmidos al nivel de los 43; 23; 20; 5,6 y 1,2 kb. Las proteínas de la membrana externa y el perfil proteico demostraron diferencias con otras especies de Shigella. Este serotipo de Shigella se aísla por primera vez en Cuba y y sus características la hacen altamente patógena y de muy difícil diagnóstico, por lo que la caracterización de este brote es importante desde el punto de vista epidemiológico

Cloned genes of the aerobactin system of virulent avian Escherichia coli do not confer virulence to recombinant strains

1. We cloned the aerobactin region and its receptor from pMV14, a large nonconjugative plasmid isolated from the virulent strain UEL14, to assess the importance of the aerobactin iron uptake system as a virulence determinant in septicemic avian Escherichia coli. 2. The physical map of the region of the recombinant plasmid (pGMV1) containing the genes for synthesis of aerobactin and its receptor was very similar to the corresponding region in pABN1 containing the genetic determinants for the aerobactin system of pColV-K30. 3. The 74-kDa outer-membrane protein encoded by pGMV1 cross-reacted immunologically with the 74-kDa aerobactin receptor protein encoded by pABN1. 4. Various avirulent E. coli strains carrying the recombinant plasmid, which contains only the aerobactin system, were assayed for virulence and were found to be avirulent for chickens. Only the wild-type aerobactin-producing strain was virulent in a pathogenicity test for chickens. 5. These results show that the aerobactin system by itself does not confer virulence, and that other factors are necessary for virulence of avian strains of E. coli

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28§C and 37§C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-Page). Several proteins with molecular weights ranging from 34 kDa to 7 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could aldso be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discuted

Haemophilus influenzae tipo b: subtipificacíon de cepas aisladas de infecciones respiratorias mediante los perfiles de proteínas de la membrana externa / Haemophilus influenzae type B: subtyping of strains isolated from respiratory infections using the outer membrane protein profiles

Se utilizó la electroforesis en un gel desnaturalizante de poliacrilamida para separar las proteínas de la membrana externa (pme) obtenidas mediante un micrométodo de extracción. Ello sirvió para subclasificar a H. influenzae b biotipo I en distintos subtipos de acuerdo a los perfiles observados. Se analizaron 37 cepas de H. influenzae b aisladas de niños con infección respiratoria aguda inferior (IRAD) y 3 de las fauces de portadores de igual sexo, edad y nivel socioeconómico, menores de cuatro años y recuperadas con la misma estacionalidad. De ellas, 27 pertenecieron al biotipo I. De acuerdo al perfil de PME, los 27 H. influenzae b biotipo I se subdividieron en 8 subtipos. La probabilidad de que dos aislamientos tomados al azar presenten diferentes subtipos de acuerdo a los perfiles de PME fue de 0.733. El subtipo que presentó mayor frecuencia fue el denominado "a", observándose tanto en las cepas invasivas como en las aisladas de fauces de los casos con IRAI y de las fauces de los niños sanos. El empleo del gel desnaturalizante de poliacrilamida al 14% permitió evidenciar la presencia de la proteína P1 con su característica de presentar distintos pesos moleculares, así como las proteínas presentes en el rango de peso molecular 25-40 KD. La mayoría de los perfiles de PME presentaron la proteína P1 de mayor peso molecular. Las condiciones de crecimiento de las cepas y la adaptación del micrométodo de extracción de PME utilizados en este estudio son procedimientos simples y al alcance de todo laboratorio clínico. Además, requiere menor insumo de tiempo que los métodos clásicos. La combinación de biotipificación, serotipificación, y determinación del perfil de PME, mostró ser un arma epidemiológicamente útil para analizar la infección a H. influenzae b.