Safety and immunogenicity of a Pseudomonas aeruginosa hybrid outer membrane protein F-I vaccine in human volunteers.
(1/1946)
A hybrid protein [Met-Ala-(His)6OprF190-342-OprI21-83] consisting of the mature outer membrane protein I (OprI) and amino acids 190 to 342 of OprF of Pseudomonas aeruginosa was expressed in Escherichia coli and purified by Ni2+ chelate-affinity chromatography. After safety and pyrogenicity evaluations in animals, four groups of eight adult human volunteers were vaccinated intramuscularly three times at 4-week intervals and revaccinated 6 months later with either 500, 100, 50, or 20 microg of OprF-OprI adsorbed onto A1(OH)3. All vaccinations were well tolerated. After the first vaccination, a significant rise of antibody titers against P. aeruginosa OprF and OprI was measured in volunteers receiving the 100- or the 500-microg dose. After the second vaccination, significant antibody titers were measured for all groups. Elevated antibody titers against OprF and OprI could still be measured 6 months after the third vaccination. The capacity of the elicited antibodies to promote complement binding and opsonization could be demonstrated by a C1q-binding assay and by the in vitro opsonophagocytic uptake of P. aeruginosa bacteria. These data support the continued development of an OprF-OprI vaccine for use in humans. (+info)
Distinct sensitivities of OmpF and PhoE porins to charged modulators.
(2/1946)
The inhibition of the anion-selective PhoE porin by ATP and of the cation-selective OmpF porin by polyamines has been previously documented. In the present study, we have extended the comparison of the inhibitor-porin pairs by investigating the effect of anions (ATP and aspartate) and positively charged polyamines (spermine and cadaverine) on both OmpF and PhoE with the patch-clamp technique, and by comparing directly the gating kinetics of the channels modulated by their respective substrates. The novel findings reported here are (1) that the activity of PhoE is completely unaffected by polyamines, and (2) that the kinetic changes induced by ATP on PhoE or polyamines on OmpF suggest different mechanisms of inhibition. ATP induces a high degree of flickering in the PhoE-mediated current and appears to behave as a blocker of ion flow during its presumed transport through PhoE. Polyamines modulate the kinetics of openings and closings of OmpF, in addition to promoting a blocker-like flickering activity. The strong correlation between sensitivity to inhibitors and ion selectivity suggests that some common molecular determinants are involved in these two properties and is in agreement with the hypothesis that polyamines bind inside the pore of cationic porins. (+info)
Enhanced bioaccumulation of heavy metal ions by bacterial cells due to surface display of short metal binding peptides.
(3/1946)
Metal binding peptides of sequences Gly-His-His-Pro-His-Gly (named HP) and Gly-Cys-Gly-Cys-Pro-Cys-Gly-Cys-Gly (named CP) were genetically engineered into LamB protein and expressed in Escherichia coli. The Cd2+-to-HP and Cd2+-to-CP stoichiometries of peptides were 1:1 and 3:1, respectively. Hybrid LamB proteins were found to be properly folded in the outer membrane of E. coli. Isolated cell envelopes of E. coli bearing newly added metal binding peptides showed an up to 1.8-fold increase in Cd2+ binding capacity. The bioaccumulation of Cd2+, Cu2+, and Zn2+ by E. coli was evaluated. Surface display of CP multiplied the ability of E. coli to bind Cd2+ from growth medium fourfold. Display of HP peptide did not contribute to an increase in the accumulation of Cu2+ and Zn2+. However, Cu2+ ceased contribution of HP for Cd2+ accumulation, probably due to the strong binding of Cu2+ to HP. Thus, considering the cooperation of cell structures with inserted peptides, the relative affinities of metal binding peptide and, for example, the cell wall to metal ion should be taken into account in the rational design of peptide sequences possessing specificity for a particular metal. (+info)
In-vitro selection of porin-deficient mutants of two strains of Klebsiella pneumoniae with reduced susceptibilities to meropenem, but not to imipenem.
(4/1946)
We have evaluated the ability of imipenem and meropenem to select, in vitro, resistant mutants of two clinical isolates of Klebsiella pneumoniae producing both SHV and TEM beta-lactamases. Only meropenem selected mutants of both isolates for which the MICs of meropenem, but not imipenem, were markedly higher than those for the parent strains; the MICs of several other beta-lactam antibiotics, including beta-lactam/beta-lactamase inhibitor combinations, for these mutants were also higher than those for the parent strains. In contrast, the MICs for the imipenem-selected mutants were the same as, or similar to, those for the parent strains. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed that an outer membrane protein in both parent strains was absent in the meropenem-selected mutants, but not in the imipenem-selected mutants. This protein is likely to be a porin, the absence of which is presumably associated with impaired beta-lactam permeability and, therefore, the reduced susceptibilities to these antibiotics exhibited by the mutant strains. We believe that this is the first report of the in-vitro selection of porin-deficient mutants of K. pneumoniae following exposure to meropenem. (+info)
Single channel analysis of recombinant major outer membrane protein porins from Chlamydia psittaci and Chlamydia pneumoniae.
(5/1946)
We recently demonstrated that the major outer membrane protein of Chlamydia psittaci, the primary vaccine candidate for combating chlamydial infections, functions as a porin-like ion channel. In this study, we have cloned, expressed and functionally reconstituted recombinant major outer membrane proteins from C. psittaci and Chlamydia pneumoniae and analysed them at the single channel level. Both form porin-like ion channels that are functionally similar to those formed by native C. psittaci major outer membrane protein. Also, like the native channels, recombinant C. psittaci channels are modified by a native major outer membrane protein-specific monoclonal antibody. This is the first time that native function has been demonstrated for recombinant chlamydial major outer membrane proteins. Future bilayer reconstitution will provide a strategy for detailed structure/function studies of this new subclass of bacterial porins and the work also has important implications for successful protein refolding and the development of improved subunit vaccines. (+info)
Site-directed mutagenesis of loop L3 of sucrose porin ScrY leads to changes in substrate selectivity.
(6/1946)
The difference in substrate selectivity of the maltodextrin (LamB) and sucrose (ScrY) porins is attributed mainly to differences in loop L3, which is supposed to constrict the lumen of the pores. We show that even a single mutation (D201Y) in loop L3 leads to a narrowing of the substrate range of ScrY to that resembling LamB. In addition, we removed the putative N-terminal coiled-coil structure of ScrY and studied the effect of this deletion on sucrose transport. (+info)
Escherichia coli outer membrane protein TolC is involved in production of the peptide antibiotic microcin J25.
(7/1946)
A Tn5 insertion in tolC eliminated microcin J25 production. The mutation had little effect on the expression of the microcin structural gene and presumably acted by blocking microcin secretion. The tolC mutants carrying multiple copies of the microcin genes were less immune to the microcin. TolC is thus likely a component of a microcin export complex containing the McjD immunity protein, an ABC exporter. (+info)
Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange.
(8/1946)
Growth factor withdrawal is associated with a metabolic arrest that can result in apoptosis. Cell death is preceded by loss of outer mitochondrial membrane integrity and cytochrome c release. These mitochondrial events appear to follow a relative increase in mitochondrial membrane potential. This change in membrane potential results from the failure of the adenine nucleotide translocator (ANT)/voltage-dependent anion channel (VDAC) complex to maintain ATP/ADP exchange. Bcl-xL expression allows growth factor-deprived cells to maintain sufficient mitochondrial ATP/ADP exchange to sustain coupled respiration. These data demonstrate that mitochondrial adenylate transport is under active regulation. Efficient exchange of ADP for ATP is promoted by Bcl-xL expression permitting oxidative phosphorylation to be regulated by cellular ATP/ADP levels and allowing mitochondria to adapt to changes in metabolic demand. (+info)