A Comparative Study of the Outer Membrane Proteome from an Atypicaland a Typical Enteropathogenic Escherichia coli

This study compared the proteomic profile of outer membrane proteins (OMPs) from one strain of atypicalenteropathogenic Escherichia coli (aEPEC) and one of typical EPEC (tEPEC). The OMPs fractions were obtained using sarcosine extraction, and analyzed by one- and two-dimensional gel electrophoresis (1DE and 2DE, respectively). The 1DE OMPs analysis of typical and atypical EPEC evidenced similar patterns; however, the 2DE OMP profile from the aEPEC revealed more protein spots in the 40- to 70-kDa region. 2DE image analysis identified 159 protein spots in bothstrains whereas 53 protein spots were observed only in tEPEC and 128 were observed only in aEPEC. Remarkably, 41.5% of aEPEC spots showed higher levels of expression compared to tEPEC, some of which with two, others four or even five times more. Twenty-four selected spots were identified using MALDI-TOF mass spectrometry and they corresponded to proteins involved in cell structure and metabolism, as well as in gene regulation. Some of these proteins showed similarity with proteins identified in other E. coli pathotypes. Besides, the differential expression of some proteins in aEPEC may suggest that it could be related to their features that ascertain the adaptation to distinct environments and the worldwidespread distribution of these pathogens.

Whole-genome analysis of Leptospira interrogans to identify potential vaccine candidates against leptospirosis

Leptospirosis is an important global human and veterinary health problem. Humans can be infected by exposure to chronically infected animals and their environment. An important focus of the current leptospiral research is the identification of outer membrane proteins (OMPs). Due to their location, leptospiral OMPs are likely to be relevant in host-pathogen interactions, hence their potential ability to stimulate heterologous immunity. The existing whole-genome sequence of Leptospira interrogans serovar Copenhageni offers a unique opportunity to search for cell surface proteins. Predicted genes encoding potential surface proteins were amplified from genomic DNA by PCR methodology and cloned into an Escherichia coli expression system. The partially purified recombinant proteins were probed by Western blotting with sera from human patients diagnosed with leptospirosis. Sixteen proteins, out of a hundred tested, were recognized by antibodies present in human sera. Four of these proteins were conserved among eight serovars of L. interrogans and absent in the non-pathogenic Leptospira biflexa. These proteins might be useful for the diagnosis of the disease as well as potential vaccine candidates.

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.