OmpA Expression Research Articles

Differential expression of ompA, ompB, pyk, nlpD and Cpn0585 genes between normal and interferon-γ treated cultures of Chlamydia pneumoniae

A common feature of many chlamydial infections is that they are often asymptomatic and may persist for long periods of time if left untreated. In addition to the well recognized lytic stage of the chlamydial developmental cycle, evidence is now emerging to support a persistent phase in the cycle in which the reticulate bodies are morphologically abnormal, viable but non-infectious and presumably also have altered gene expression patterns. We used an RT-PCR approach to study the differential levels of gene transcription for 14 genes (16SrRNA, ompA, ompB, omcB, 76 kDa, gseA, pmp1, gltX, hsp60, yaeT, pyk, nlpD, Cpn0585, Cpn1046) between normal and IFN-γ treated Chlamydia pneumoniae cell cultures. Even though the level of morphologically abnormal reticulate bodies in our IFN-γ treated cultures was low (approximately 10% morphologically discernible, although presumably a larger percentage were in the persistent state but not yet morphologically altered) we identified five genes (ompA, ompB, pyk, nlpD, Cpn0585) that were clearly upregulated when compared to normal cultures. This gene transcript profile may be characteristic of a general stress state in Chlamydia, induced by IFN-γ treatment in this case, but perhaps more widely induced in other in vitro and in vivo situations.

Hfq (HF1) stimulates ompA mRNA decay by interfering with ribosome binding

The adaptation of mRNA stability to environmental changes is a means of cells to adjust the level of gene expression. The Escherichia coli ompA mRNA has served as one of the paradigms for regulated mRNA decay in prokaryotes. The stability of the transcript is known to be correlated inversely with the bacterial growth rate. Thus, the regulation of ompA mRNA stability meets the physiological needs to adjust the level of ompA expression to the rate of cell division. Recently, host factor I (Hfq/HF1) was shown to be involved in the regulation of ompA mRNA stability under slow growth conditions. Here, we present the first direct demonstration that 30S ribosomes bound to the ompA 5'-UTR protect the transcript from RNase E cleavage in vitro. However, the 30S protection was found to be abrogated in the presence of Hfq. Toeprinting and in vitro translation assays revealed that translation of ompA is repressed in the presence of Hfq. These in vitro studies are corroborated by in vivo expression studies demonstrating that the reduced synthesis rate of OmpA effected by Hfq results in functional inactivation of the ompA mRNA. The data are discussed in terms of a model wherein Hfq regulates the stability of ompA mRNA by competing with 30S ribosomes for binding to the ompA 5'-UTR.

Outer membrane protein A-promoted actin condensation of brain microvascular endothelial cells is required for Escherichia coli invasion.

Escherichia coli is the most common gram-negative bacterium that causes meningitis during the neonatal period. We have previously shown that the entry of circulating E. coli organisms into the central nervous system is due to their ability to invade the blood-brain barrier, which is composed of a layer of brain microvascular endothelial cells (BMEC). In this report, we show by transmission electron microscopy that E. coli transmigrates through BMEC in an enclosed vacuole without intracellular multiplication. The microfilament-disrupting agents cytochalasin D and latrunculin A completely blocked E. coli invasion of BMEC. Cells treated with the microtubule inhibitors nocodazole, colchicine, vincristin, and vinblastine and the microtubule-stabilizing agent taxol also exhibited 50 to 60% inhibition of E. coli invasion. Confocal laser scanning fluorescence microscopy showed F-actin condensation associated with the invasive E. coli but no alterations in microtubule distribution. These results suggest that E. coli uses a microfilament-dependent phagocytosis-like endocytic mechanism for invasion of BMEC. Previously we showed that OmpA expression significantly enhances the E. coli invasion of BMEC. We therefore examined whether OmpA expression is related to the recruitment of F-actin. OmpA(+) E. coli induced the accumulation of actin in BMEC to a level similar to that induced by the parental strain, whereas OmpA(-) E. coli did not. Despite the presence of OmpA, a noninvasive E. coli isolate, however, did not show F-actin condensation. OmpA(+)-E. coli-associated condensation of F-actin was blocked by synthetic peptides corresponding to the N-terminal extracellular domains of OmpA as well as BMEC receptor analogues for OmpA, chitooligomers (GlcNAcbeta1-4GlcNAc oligomers). These findings suggest that OmpA interaction is critical for the expression or modulation of other bacterial proteins that will subsequently cause actin accumulation for the uptake of bacteria.

Characterization of Escherichia coli expressing an Lpp'OmpA(46-159)-PhoA fusion protein localized in the outer membrane.

The Lpp'OmpA(46-159) hybrid protein can serve as an efficient targeting vehicle for localizing a variety of procaryotic and eucaryotic soluble proteins onto the E. coli surface, thus providing a system for several possible biotechnology applications. Here we show that fusion between Lpp'OmpA(46-159) and bacterial alkaline phosphatase (PhoA), a normally periplasmic dimeric enzyme, are also targeted to the outer membrane. However, protease accessibility experiments and immunoelectron microscopy revealed that, unlike other periplasmic proteins, the PhoA domain of these fusions is not exposed on the cell surface in cells having an intact outer membrane. Conditions that affect the formation of disulfide bonds and the folding of the PhoA domain in the periplasm not only did not facilitate targeting to the cell surface but led to lethality when the fusion was expressed from a high-copy-number plasmid. Furthermore, E. coli expressing the Lpp'OmpA(46-159)-PhoA fusion exhibited strain- and temperature-dependent alterations in outer-membrane permeability. Our results are consistent with previous studies with other vehicles indicating that PhoA is not displayed on the surface when fused to cell-surface expression vectors. Presumably, the enzyme rapidly assumes a tightly folded dimeric conformation that cannot be transported across the outer membrane. The large size and quaternary structure of PhoA may define a limitation of the Lpp'OmpA(46-159) fusion system for the display of periplasmic proteins on the cell surface. Alkaline phosphatase is a unique protein among a group of five periplasmic proteins (beta-lactamase, alkaline phosphatase, Cex cellulase Cex cellulose-binding domain, and a single-chain Fv antibody fragment), which have been tested as passengers for the Lpp'OmpA(46-159) expression system to date, since it was the only protein not displayed on the surface.

Secretion of eukaryotic growth hormones in Escherichia coli is influenced by the sequence of the mature proteins

We report the construction of secretion plasmids expressing the fusion proteins, OmpA::pGH (pSpGH.01) and OmpA::hGH (phGH.01), and compare the secretion of mature porcine growth hormone (pGH) and human growth hormone (hGH) employing Escherichia coli. E. coli [phGH.O1] secreted 10–15 μg hGH/m1 A 100 cells into the periplasmic space, representing 30% of total periplasmic proteins. E. coli [pSpGH.01], however, secreted 30-fold less mature pGH. On the basis that both pSpGH.O1 and phGH.O1 are stably maintained in E. coli and in vitro transcription/translation data showed equivalent expression of OmpA::pGH and OmpA::hGH precursors, we attribute the higher secretion of hGH to the translocation-competent OmpA::hGH protein configuration. Two OmpA::GHF (growth hormone fusion) precursors, OmpA::GHF.02 and OmpA::GHF.03, both with hGH helix 3/helix 4 together instead of the pGH equivalent, secreted mature proteins as efficiently as OmpA::hGH. We propose that hGH helices 3 and 4 in these OmpA::GHF precursors play a major role in the folding of the precursor to a translocation-competent state, mimicking the translocation-competent nature of the OmpA::hGH precursor.

Cloning, expression and characterization of human kininogen domain 3

The internal domain 3 of the heavy chain of human kininogen, a cysteine proteinase inhibitor, was amplified by a polymerase chain reaction from the kininogen cDNA clone phKG36. The DNA fragment was expressed in Escherichia coli using the ompA expression vector pASK.40 and the resulting protein was isolated from periplasm, purified by S-carboxymethylpapain affinity- and ion-exchange chromatography. The recombinant human kininogen domain 3 is 92% pure, reacts with anti-kininogen antibodies and is actively inhibitory. The expected amino acid sequence of ANSM-[G253-S377] kininogen was confirmed; the inhibitor has a molecular mass of 14,396 Da and an isoelectric point of 6.0 (pH). The determined K i values of the complexes with papain and cathepsin L are similar to those measured previously with proteolytically liberated kininogen domain 3, and those of single-domain cystatins, like chicken egg white cystatin. However, recombinant kininogen domain 3 is a weak inhibitor of cathepsin B ( K i = 63 nM) as it has been found for native L-kininogen ( K i = 340 nM).

Molecular characterization of an Enterobacter cloacae gene (romA) which pleiotropically inhibits the expression of Escherichia coli outer membrane proteins

The introduction of a newly cloned Enterobacter cloacae chromosomal gene romA, into Escherichia coli and E. cloacae resulted in enhancement of resistance to quinolones, beta-lactams, chloramphenicol, and tetracycline. The primary effect of romA on a multicopy vector in E. coli was almost complete inhibition of OmpF expression in the outer membrane. From the experiments with ompR and envZ mutants or with ompF-lacZ and ompC-lacZ fusion plasmids, it was concluded that this inhibition is posttranscriptional. The introduction of romA on a multicopy vector into strains with micF deletion elicited only a moderate decrease in OmpF protein expression. This indicates that reduction of OmpF expression by romA is partly mediated posttranscriptionally by the activation of micF. Moreover, the overexpression of RomA protein from an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter resulted in nearly complete inhibition of expression of OmpC and OmpA, as well as OmpF. Taken together with an observation in a recent study that overexpressed OmpC inhibited the synthesis of OmpA and LamB, a possible inhibitory mechanism at the translational stage of the synthesis of outer membrane proteins should also be considered. By Southern hybridization, romA was generally detected in the chromosomes of all E. cloacae strains tested but not in the E. coli K-12 chromosome. Sequence data show that there is an open reading frame specifying 368 amino acids residues including a putative signal peptide. RomA appears to belong to the outer membrane protein family since it was extractable from an outer membrane preparation, but no sequence homology to other outer membrane proteins was detected.

The influence of amino substitutions within the mature part of an Escherichia coli outer membrane protein (OmpA) on assembly of the polypeptide into its membrane.

The membrane part of the 325-residue outer membrane protein OmpA of Escherichia coli encompasses residues 1-177. This part is thought to cross the membrane eight times in antiparallel beta-strands, forming four loops of an amphipathic beta-barrel. With the aim of gaining some insight into the mechanism of sorting, i.e. the way the protein recognizes and assembles into its membrane, a set of point mutants in the ompA gene has been generated. Selection for toxicity of ompA expression following mutagenesis with sodium bisulfite yielded genes with multiple base pair substitutions, the majority of which resulted in amino acid substitutions in the membrane moiety of the protein. None of the altered proteins was blocked in membrane incorporation. A proline residue exists at or near each of the presumed turns at the inner side of the outer membrane. Using oligonucleotide-directed mutagenesis, each of them was replaced by a leucine residue which is thought to be a turn blocking residue. None of these proteins had lost the ability to be incorporated into the membrane. Apparently, leucine residues are tolerated at turns in this protein. To interfere with the formation of antiparallel beta-strands, four double mutants were prepared: ompA-ON3 (Ala11----Pro, Leu13----Pro), -ON4 (Ala11----Asp, Leu13----Pro), -ON5 (Gly160----Val, Leu162----Arg), and -ON6 (Leu164----Pro, Val166----Asp). The former three proteins and even quadruple mutants consisting of a combination of ompA-ON2 or -ON4 with -ON5 were not defective in membrane assembly. In contrast, the OmpA-ON6 protein was translocated across the plasma membrane but could not be incorporated into the outer membrane. It is concluded that at least one rather small area of the polypeptide is of crucial importance for the assembly of OmpA into the outer membrane.

Roles of the 5′ leader region of the ompA mRNA

OmpA protein, a major outer membrane protein of Escherichia coli, is synthesized from a messenger RNA containing a 134-nucleotide 5′ leader region. The role of this leader region in efficient ompA expression was investigated using a series of ompA- lacZ fusion plasmids. These plasmids differ in the amount of DNA encoding the ompA leader region which is fused to the lacZ structural gene. The fusion containing all but six nucleotides of the ompA leader produced the highest β-galactosidase activity, while the fusion containing the shortest leader synthesized only 4% as much β-galactosidase. Fusions with leaders intermediate in length produced between 6% and 24% of the activity found in the most efficient fusion. Quantitaten of lacZ mRNA synthesis by DNA-RNA hybridization revealed differences in lacZ mRNA production reflecting the observed differences in β-galactosidase activity. The primary effect of the ompA leader in maintaining high levels of mRNA is discussed in terms of the roles of mRNA secondary structure.