Chimeric animal and plant viruses expressing epitopes of outer membrane protein F as a combined vaccine against Pseudomonas aeruginosa lung infection

Outer membrane protein F of Pseudomonas aeruginosa has vaccine efficacy against infection by P. aeruginosa as demonstrated in a variety of animal models. Through the use of synthetic peptides, three surface-exposed epitopes have been identified. These are called peptides 9 (aa 261-274 in the mature F protein, TDAYNQKLSERRAN), 10 (aa 305-318, NATAEGRAINRRVE), and 18 (aa 282-295, NEYGVEGGRVNAVG). Both the peptide 9 and 10 epitopes are protective when administered as a vaccine. In order to develop a vaccine that is suitable for use in humans, including infants with cystic fibrosis, the use of viral vector systems to present the protective epitopes has been investigated. An 11-amino acid portion of epitope 10 (AEGRAINRRVE) was successfully inserted into the antigenic B site of the hemagglutinin on the surface of influenza virus. This chimeric influenza virus protects against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Attempts to derive a chimeric influenza virus carrying epitope 9 have been unsuccessful. A chimeric plant virus, cowpea mosaic virus (CPMV), with epitopes 18 and 10 expressed in tandem on the large coat protein subunit (CPMV-PAE5) was found to elicit antibodies that reacted exclusively with the 10 epitope and not with epitope 18. Use of this chimeric virus as a vaccine afforded protection against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Chimeric CPMVs with a single peptide containing epitopes 9 and 18 expressed on either of the coat proteins are in the process of being evaluated. Epitope 9 was successfully expressed on the coat protein of tobacco mosaic virus (TMV), and this chimeric virus is protective when used as a vaccine in the mouse model of chronic pulmonary infection. However, initial attempts to express epitope 10 on the coat protein of TMV have been unsuccessful. Efforts are continuing to construct chimeric viruses that express both the 9 and 10 epitopes in the same virus vector system. Ideally, the use of a vaccine containing two epitopes of protein F is desirable in order to greatly reduce the likelihood of selecting a variant of P. aeruginosa that escapes protective antibodies in immunized humans via a mutation in a single epitope within protein F. When the chimeric influenza virus containing epitope 10 and the chimeric TMV containing epitope 9 were given together as a combined vaccine, the immunized mice produced antibodies directed toward both epitopes 9 and 10. The combined vaccine afforded protection against challenge with P. aeruginosa in the chronic pulmonary infection model at approximately the same level of efficacy as provided by the individual chimeric virus vaccines. These results prove in principle that a combined chimeric viral vaccine presenting both epitopes 9 and 10 of protein F has vaccine potential warranting continued development into a vaccine for use in humans.     

Reactivity of elementary and reticulate bodies of Chlamydia trachomatis LGV2 with monoclonal antibodies specific for the major outer membrane protein

The reactivity of the major outer membrane protein (MOMP) of Chlamydia trachomatis (LGV2 serotype) with 15 monoclonal antibodies was studied during the course of developmental cycle by immunoblotting and immunofluorescence. The monoclonal antibodies reacted in immunoblots with the MOMP of both elementary bodies (EBs) and reticulate bodies (RBs). Using an immunofluorescence test with LGV2-infected cell cultures, the 15 monoclonal antibodies could be divided into 5 groups, according to the time of appearance of their reactivity with the cell culture.     

Exchange of colicin receptor capacity between strains of Escherichia coli sensitive or resistant to colicin K-K235

Phage and colicin-resistant mutants were derived from Escherichia coli K-12P678. Two classes of phage T6 and colicin K-resistant mutants (genotype tsx) were isolated. Tsx-2 mutants, which demonstrated mucoid growth and increased sensitivities to many antibiotics, became sensitive to colicin K when pretreated with ethylenediaminetetraacetate (EDTA), whereas Tsx-1 mutants did not. Reassociation of EDTA-released material partially restored resistance to colicin K for Tsx-2 mutants. When EDTA-released material from strain P678 was associated with either class of K-resistant mutant, an increase in colicin K sensitivity resulted. Observations suggest that colicin K can act on its target site once it penetrates the cell surface. In addition, results suggest that functional colicin K receptors can be transferred from sensitive to resistant strains, thus conferring colicin sensitivity.Non-standard Abbreviations SDS sodium dodecyl sulfate     

A disulfide bridge network within the soluble periplasmic domain determines structure and function of the outer membrane protein RCSF

RcsF, a proposed auxiliary regulator of the regulation of capsule synthesis (rcs) phosphorelay system, is a key element for understanding the RcsC-D-A/B signaling cascade, which is responsible for the regulation of more than 100 genes and is involved in cell division, motility, biofilm formation, and virulence. The RcsC-D-A/B system is one of the most complex bacterial signal transduction pathways, consisting of several membrane-bound and soluble proteins. RcsF is a lipoprotein attached to the outer membrane and plays an important role in activating the RcsC-d-A/B pathway. The exact mechanism of activation of the rcs phosphorelay by RcsF, however, remains unknown. We have analyzed the sequence of RcsF and identified three structural elements: 1) an N-terminal membrane-anchored helix (residues 3-13), 2) a loop (residues 14-48), and 3) a C-terminal folded domain (residues 49-134). We have determined the structure of this C-terminal domain and started to investigate its interaction with potential partners. Important features of its structure are two disulfide bridges between Cys-74 and Cys-118 and between Cys-109 and Cys-124. To evaluate the importance of this RcsF disulfide bridge network in vivo, we have examined the ability of the full-length protein and of specific Cys mutants to initiate the rcs signaling cascade. The results indicate that the Cys-74/Cys-118 and the Cys-109/Cys-124 residues correlate pairwise with the activity of RcsF. Interaction studies showed a weak interaction with an RNA hairpin. However, no interaction could be detected with reagents that are believed to activate the rcs phosphorelay, such as lysozyme, glucose, or Zn(2+) ions.     

Isolation and characterization of mutants deleted for the sulA-ompA region of the Escherichia coli K-12 chromosome

Abstract We have isolated mutants deleted for different segments of the sulA-ompA region of the Escherichia coli K-12 chromosome using gene fusion techniques. Genetic and physical analysis showed that the deletions ranged from 500 to more than 4000 base pairs (bp) Strains were found in which all, or part, of the sulA and ompA genes had been deleted.     

Elektronenmikroskopische Untersuchungen am Pinealorgan von Pterophyllum scalare Cuv. et Val. (Cichlidae,Teleostei)

Zusammenfassung Das Pinealorgan (Epiphysis cerebri) des Knochenfisches Pterophyllum scalare besteht aus nervösen und gliösen Zellelementen. Sehr stark ausgebildet sind die ependymalen Stützzellen. Sie umhüllen mit ihren Ausläufern, die sich überlappen können, andere Zellelemente, z.B. Rezeptorzellen und marklose Nervenfasern. Neben dieser Neuroglia-Art finden sich auch noch oligodendrocytenähnliche Gliazellen. In ihrer Grundstruktur entsprechen die Rezeptoren den Epiphysensinneszellen anderer Knochenfische. Vom cilientragenden Teil des Außenglieds geht ein schürzenartiger Lamellenstapel aus. Dieser besteht aus 50–70 Lamellenplatten von etwa 6 m Länge. Im basalen Teil der Rezeptorzelle sind neben schlanken Mitochondrien mit unregelmäßigen Cristae und Tubuli auch noch große, runde Mitochondrien mit einer regelmäßigen Cristastruktur zu beobachten. Der basale Fortsatz der Rezeptorzelle ist auf die axial verlaufenden Axonbündel ausgerichtet. Synapsenartige Kontakte sind selten. Die Zahl der marklosen Axone nimmt hirnwärts zu; dieser Befund wurde in partiellen Rekonstruktionen gesichert. Am Übergang in den Epiphysenstiel treten einige markhaltige Axone auf. Zur Verteilung der Zelltypen und zum Verlauf der Axonbündel im Epiphysenstiel des Skalars liegen detailliertere Angaben vor als bei anderen bisher untersuchten Knochenfischepiphysen. In der Diskussion werden nach Vergleich der pinealen Rezeptoren verschiedener Fische drei Außengliedformen unterschieden: Bürsten-, Schürzen- und Kappentyp. Diese Varianten werden in Verbindung mit den bekannten physiologischen Reaktionsformen der Pinealorgane diskutiert. Die elektrophysio logischen Unterschiede lassen sich nicht mit verschiedenen Strukturtypen des Außenglieds erklären.

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Electron microscopic studies of the pineal organ in Pterophyllum scalare cuv. et val. (Cichlidae, Teleostei)

Summary The pineal organ (epiphysis cerebri) of Pterophyllum scalare is formed by neuronal and glial elements. Ependymal supportive cells are very abundant, and their cytoplasmic processes envelop adjacent receptor cells and unmyelinated nerve fibers by an intertwining network. In addition to this type of neuroglia, oligodendrocytic cells have also been identified. The receptor cells show the general structural pattern (outer segment, inner segment, basal process) of teleostean pineal receptors. The ciliary part of the outer segment bears a dome-like stack of 50–70 curved saccules each of average length of 6 m. In the basal part of the receptor cell, slender mitochondria containing irregular cristae and tubules, and also some more spherical mitochondria with a highly regular arrangement of cristae, can be observed. The basal cytoplasmic process radiates into neuropile-like areas that contain axial bundles of axons. Synaptoid contacts rarely occur. The number of unmyelinated axons of the pineal stalk, increases in a proximad direction (towards the brain). This finding has been verified in partial reconstructions. In the transitional zone leading from the pineal body into the pineal stalk, a few myelinated fibers become visible. With respect to cell types and the axonic bundles of the pineal stalk in Pterophyllum scalare, more detailed data are presented than for most other teleostean pineal organs examined thus far. The comparison of pineal sensory cells in several fishes allows a distinction among three different types of outer segments, i.e., a slender type, a dome-like type, and a cap-like type. These structural types are discussed with respect to the relevant physiological results. The existence of particular structural types of the outer segment does not explain the different electrophysiological reactions observed in different pineal organs.

Ein Druckkostenzuschuß wurde von beiden Instituten zur Verfügung gestellt.     

O-Serogrouping and surface components of Aeromonas hydrophila and Aeromonas jandaei pathogenic for eels

Abstract The relationship between virulence, O-serogroup, and some cell-surface features (self-pelleting [SP] and precipitation after boiling [PAB], profile of lipopolysaccharides [LPSs]) and outer membrane proteins [OMPs] was investigated in strains of the pathogenic species Aeromonas hydrophila and A. jandaei isolated from eels. Virulent strains of A. hydrophila reacted mostly with O:19 antiserum, and those of A. jandaei reacted with O:4, O:11, O:15 and O:29 antisera (Guinée and Jansen system). Regarding the PAB and LPS profiles two groups could be distinguished; (i) five PAB⁺ strains of serotype O:19 that possessed a homogeneous O polysaccharide side chain and (ii) thirteen PAB⁻ strains antigenically diverse that either exhibited a heterogenous side chain or were side chain deficient. A major 50 kDa protein was only found in the PAB⁺ strains, whereas major OMPs detected in PAB⁻ strains ranged from 33 to 45 kDa irrespective of the species. Epizootic eel isolates of A. hydrophila belong to serotype O:19 and share cell-surface features with the Aeromonas highly virulent for other hosts. In contrast, epizootic A. jandaei isolates were antigenically diverse. These findings reinforce the importance of an O-serotype as an epidemiological marker in motile Aeromonas strains pathogenic for eels.