A role for alpha-and beta-catenins in bacterial uptake.

Interaction of internalin with E-cadherin promotes entry of Listeria monocytogenes into human epithelial cells. This process requires actin cytoskeleton rearrangements. Here we show, by using a series of stably transfected cell lines expressing E-cadherin variants, that the ectodomain of E-cadherin is sufficient for bacterial adherence and that the intracytoplasmic domain is required for entry. The critical cytoplasmic region was further mapped to the beta-catenin binding domain. Because beta-catenin is known to interact with alpha-catenin, which binds to actin, we generated a fusion molecule consisting of the ectodomain of E-cadherin and the actin binding site of alpha-catenin. Cells expressing this chimera were as permissive as E-cadherin-expressing cells. In agreement with these data, alpha- and beta-catenins as well as E-cadherin clustered and colocalized at the entry site, where F-actin then accumulated. Taken together, these results reveal that E-cadherin, via beta- and alpha-catenins, can trigger dynamic events of actin polymerization and membrane extensions culminating in bacterial uptake.

Temperature sensing in bacterial gene regulation--what it all boils down to.

Many bacterial gene regulatory circuits are controlled by temperature. Temperature-mediated regulation occurs at the level of transcription and translation. Supercoiling, changes in mRNA conformation and protein conformation are all implicated in thermosensing. Bacterial virulence functions are often temperature regulated and thus many an example of thermoregulation comes from pathogenic organisms. H-NS is at the crossroads of regulation in many such systems. mRNA melting has also been shown to act as a thermosensing mechanism in various contexts. Proteins can also act as temperature sensors as exemplified by the gene regulator TlpA in Salmonella typhimurium.

A proteinaceous gene regulatory thermometer in Salmonella.

Novel utilization of the coiled-coil motif is presented that enables TlpA, an autoregulatory repressor protein in Salmonella, to sense temperature shifts directly and thereby to modulate the extent of transcription repression. Salmonella cells shifted to higher temperatures, such as those encountered at host entry, showed derepressed tlpA activity. tlpA::lacZ fusions indicated that the promoter itself is insensitive to thermal shifts and that transcription control was exerted by the autorepressor TlpA only. In vitro studies with highly purified TlpA showed concentration and temperature dependence for both fully folded conformation and function, indicating that the thermosensing in TlpA is based on monomer-to-coiled-coil equilibrium.

DNA binding exerted by a bacterial gene regulator with an extensive coiled-coil domain.

Although quite common in the eukaryotic cell, bacterial proteins with an extensive coiled-coil domain are still relatively rare. One of the few thus far documented examples, TlpA from Salmonella typhimurium, is characterized by a remarkably long (250 amino acids) alpha-helical coiled-coil domain. Herein, we demonstrate that TlpA is a novel sequence-specific DNA-binding protein. Several tlpA deletion mutants have been constructed, and their corresponding protein products were purified and tested for DNA binding. Two of the mutant proteins were shown to be deficient in DNA binding. Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins wre shown to be deficient in DNA binding. Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins were largely intact despite lacking the amino acid residues necessary for DNA binding. In vivo studies with transcriptional tlpA-lacZ fusions demonstrated that TlpA acts as a repressor. Using the repressor phenotype as a readout, the chain exchange previously described in vitro could also be confirmed in vivo. We believe the coiled-coil domain acts not only as a dimerization interface but could also serve a role as a flexible modulator of the protein-DNA interaction.

Evidence for functional polymorphism of the spvR gene regulating virulence gene expression in Salmonella.

The expression of Salmonella enterica spv virulence genes was studied in serovariants Dublin and Typhimurium using Western blotting (immunoblotting), spv-lacZ operon fusions and Northern blotting. The SpvA protein was detected in immunoblots from stationary phase cultures of Dublin but not from the corresponding cultures of Typhimurium. Transcriptional measurements, using a spvA-lacZ operon fusion, indicated 8-10 times higher spvA transcription in Dublin. In an isogenic Escherichia coli chromosomal background, virulence plasmids from various Dublin strains systematically had a significantly higher induction level of the spvA-lacZ operon fusion than virulence plasmids from Typhimurium strains. The cloned spvR transcriptional activator gene of Dublin strain 2229 was found to activate both spvR-lacZ and spvA-lacZ operon fusions, as well as to raise spv mRNA levels in E. coli TG1. In contrast, the corresponding cloned gene of Typhimurium strain SL2965 possessed a lower induction potential and required higher spvR gene dosage for activation. A comparison of the nucleotide sequences of spvR genes from two Dublin and four Typhimurium strains revealed conserved, serovariant-associated basepair substitutions. Our results indicate that the spv virulence gene cluster possesses different functional alleles of the regulator gene spvR. This finding has important consequences for comparative studies of regulation and virulence in different serovariants of Salmonella.

Intermediate filament-like network formed in vitro by a bacterial coiled coil protein.

The TlpA protein encoded by the virulence plasmid of Salmonella enterica is an alpha-helical 371-amino acid protein possessing characteristics similar to eukaryotic coiled coil proteins (Koski, P., Saarilahti, H., Sukupolvi, S., Taira, S., Rikkonen, P., Osterlund, K., Hurme, R., and Rhen, M. (1992) J. Biol. Chem. 267, 12258-12265). In this paper we have investigated inter- and intramolecular associations and the morphology of structures formed by TlpA. Dynamics and temperature stability of TlpA dimers were studied by examining the feasibility and conditions in which TlpA would form an artificial heterodimer with its truncated derivative. Formation of heterodimers, bridged by Cu(2+)-catalyzed air oxidation of adjacent Cys residues, showed that TlpA dimers are dynamic chain exchanging structures at 37 degrees C, whereas they were nonexchanging at room temperature or on ice. Chemical cross-linking suggested higher order interaction between TlpA dimers. Electron microscopy studies revealed two levels of TlpA organization in vitro: thin filaments and rods, 2-5 nm in diameter, and a higher ordered filament network consisting of tonofilament-like formations with a diameter of 8-15 nm. Electron microscopy of thin-sectioned Escherichia coli over-producing TlpA showed an extraordinary intracellular assembly of proteinacious lamellae with a striated appearance and a 38-nm periodicity. This study describes for the first time a bacterial protein capable of organizing itself into an ordered and suspectedly dynamic intermediate filament-like architecture.

A new alpha-helical coiled coil protein encoded by the Salmonella typhimurium virulence plasmid.

A new protein of Salmonella typhimurium was identified and characterized. The gene (tlpA) encoding this protein (TlpA) was isolated from the large virulence-associated plasmid of S. typhimurium and sequenced in order to predict the primary structure of TlpA. tlpA encodes a 371-amino acid soluble protein with a calculated M(r) of 41600 and pI of 4.63. Secondary structure predictions and sequence statistics of TlpA indicated a predominant alpha-helical configuration and presence of heptapeptide repeat motifs characteristic of coiled coil proteins. Purified TlpA was shown to have biochemical properties similar to those of coiled coil proteins, including adoption of an alpha-helical configuration and a tendency to form homodimers. Furthermore, TlpA possessed heat resistance, evidence for a chain register and altered mobility in urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels which are characteristics of tropomyosins. TlpA shows 32% overall sequence similarity with rat cardiac myosin and 36% similarity with horse platelet beta-tropomyosin over 226 residues, whereas selected regions possessed significant sequence identities with myosins, tropomyosins, and alpha-helical surface proteins of Streptococcus pyogenes. Our results indicate that TlpA represents a new member of prokaryotic coiled coil proteins.

Separation and characterization of two chemically distinct lipopolysaccharides in two Pectinatus species.

Lipopolysaccharides (LPS) from the type strains of the anaerobic beer spoilage bacteria Pectinatus cerevisiiphilus and P. frisingensis were extracted with the 5:5:8 volume ratio modification of the phenolchloroform-petroleum ether method (H. Brade and C. Galanos, Eur. J. Biochem. 122:233-237, 1982). Sequential precipitations of LPS with water and acetone from the phenol phase yielded LPS which differed in that water-precipitable material (LPS-H2O; 0.1 to 0.4% of the dry weight of the cells) was rough-type LPS, whereas acetone-precipitable material (LPS-Ac; 4.6 to 5.8% of the dry weight) contained both rough-type LPS and high-molecular-weight material resembling smooth LPS. The LPS were chemically characterized, and they contained D-glucosamine, 4-amino-4-deoxy-L-arabinose, 3-deoxy-D-manno-2-octulosonic acid, D-fucose, D-galactose, D-glucose, D-mannose, and phosphate. D-Fucose was present mostly in LPS-Ac, suggesting that it is a constituent of the O antigen. The major fatty acids were ester- and amide-linked (R)-3-hydroxytridecanoic and ester-linked undecanoic acids, with minor amounts of ester-linked tridecanoic and (R)-3-hydroxyundecanoic acids. The chemical compositions of LPS-H2O and LPS-Ac suggested that they differ not only in their smooth or rough nature but also in the structure of their core regions. This may explain their different precipitabilities from the extraction mixture. The extraction method was also shown to be applicable to the isolation of smooth-type LPS from Salmonella enterica serovar Typhimurium. Extraction of two Typhimurium strains carrying chemically different O antigens resulted in high yields (8% of the dry weight) of LPS. Strain SH2183, which contains the relatively hydrophobic O-4,5,12 antigen yielded almost exclusively LPS-Ac, whereas the LPS of strain SH5770, which has a hydrophilic O-6,7 antigen, was exclusively LPS-H2O. No fractionation to smooth and rough LPS occurred with the Typhimurium strains.

Comparison of ketotifen, disodium cromoglycate and placebo in the treatment of adult patients with mild extrinsic asthma.

Sixty-four patients with mild of moderate extrinsic asthma were treated with placebo for 1 month and thereafter with ketotifen (1 mg twice daily, orally), disodium cromoglycate (inhalation of 20 mg, four times daily), or placebo for 2 subsequent months. The trial was performed at four different centres and the treatments were compared using double-blind technique. We found no difference between the effect of ketotifen, disodium cromoglycate and placebo on the patients' daily measurements of evening peak expiratory flow, daily score values or respiratory symptoms of the number of salbutamol puffs required to control symptoms. There was no difference between the treatment groups with regard to the patients' estimates of changes in airway sensitivity to different non-specific stimuli: fumes, tobacco smoke, cold air, and exercise. The only significant effect of DSCG was a minor (4%) increase in the mean morning value for peak expiratory flow. The findings suggest that the addition of ketotifen or disodium cromoglycate to the regimen is unlikely to give further benefit in asthmatic patients, whose symptoms are reasonably well controlled by small doses of bronchodilating drugs.

A controlled study on the preventive effect of ketotifen, an antiallergic agent, on methacholine-induced bronchoconstriction in asthmatics.

We studied the preventive effect of ketotifen, an oral drug with antianaphylactic and antihistaminic properties on methacholine-induced bronchoconstriction in controlled cross-over experiments in twenty-six adult patients with extrinsic asthma. Both a single dose of 1 mg ketotifen and 4 weeks treatment of ketotifen, 1 mg twice daily, failed to reduce the methacholine-induced drop in peak expiratory flow. The spirometric findings remained unchanged during ketotifen treatment. There was no difference between treatments with ketotifen and placebo with regard to the patients assessment of the severity of asthma or airway sensitivity to tobacco smoke, fumes or dusts, or exercise. The results suggest that treatment during 4 weeks with ketotifen does not reduce unspecific broncial hyperreactivity in patients with extrinsic asthma.

Preventive effect of ketotifen, a new antiallergic agent, on histamine-induced bronchoconstriction in asthmatics.

The preventive effect of ketotifen, a new drug with anti-histaminic and antiallergic properties, on histamine-induced bronchoconstriction was studied by open assessment in twenty-four adult patients with extrinsic asthma. A single oral dose of 1 mg ketotifen reduced the post-histamine mean drop in peak expiratory flow from 33 to 16% of the basal values (P less than 0.001). After a 4 weeks' regimen of 1 mg ketotifen twice daily the post-drug histamine-induced fall in PEF was further significantly reduced (P less than 0.001). Tests performed after 8 and 12 weeks of treatment showed no additional decrease in bronchial reactivity to histamine. Tests performed 1 week after cessation of treatment showed return of bronchial reactivity to the pretreatment level. The results suggest that a single dose of ketotifen has a marked preventive effect on histamine-induced bronchoconstriction and that this effect is enhanced during continued treatment.

Treatment of acute bronchitis and pneumonia with cefaclor.

An open, non-comparative clinical trial of cefaclor in adults with acute exacerbations of chronic bronchitis (54 patients) or pneumonia (24 patients) is reported. The dosage of cefaclor used was either 250 mg or 500 mg taken orally three times daily. Clinical cure was obtained in 39 of 42 (93%) of patients on the lower dose and 32 of 33 (97%) on the higher dose. Side effects were minimal and the antibiotic was very well tolerated. Microbiological evaluation was possible in 32 of 75 (43%) of patients in whom potential pathogens were identified before treatment. Microbiological 'cure' was achieved in 17 of 32 (53%), the majority of whom had received the higher dose. The lack of correlation between the clinical and microbiological results cast further doubts on the value of the standard sputum culture methods in the diagnosis and management of lower respiratory tract infection. Cefaclor is useful in the management of acute lower respiratory tract infections by virtue of its excellent clinical efficacy and safety.