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Jana Jass

Publications and source records attributed to Jana Jass.

14 recordsLinked to original sources
PubMed2005

The unfolding of the P pili quaternary structure by stretching is reversible, not plastic.

P pili are protein filaments expressed by uropathogenic Escherichia coli that mediate binding to glycolipids on epithelial cell surfaces, which is a prerequisite for bacterial infection. When a bacterium, attached to a cell surface, is exposed to external forces, the pili, which are composed of approximately 10(3) PapA protein subunits arranged in a helical conformation, can elongate by unfolding to a linear conformation. This property is considered important for the ability of a bacterium to withstand shear forces caused by urine flow. It has hitherto been assumed that this elongation is plastic, thus constituting a permanent conformational deformation. We demonstrate, using optical tweezers, that this is not the case; the unfolding of the helical structure to a linear conformation is fully reversible. It is surmised that this reversibility helps the bacteria regain close contact to the host cells after exposure to significant shear forces, which is believed to facilitate their colonization.

Escherichia coli↗
PubMed2004

Physical properties of Escherichia coli P pili measured by optical tweezers.

The mechanical behavior of individual P pili of uropathogenic Escherichia coli has been investigated using optical tweezers. P pili, whose main part constitutes the PapA rod, composed of approximately 10(3) PapA subunits in a helical arrangement, are distributed over the bacterial surface and mediate adhesion to host cells. They are particularly important in the pathogenesis of E. coli colonizing the upper urinary tract and kidneys. A biological model system has been established for in situ measurements of the forces that occur during mechanical stretching of pili. A mathematical model of the force-versus-elongation behavior of an individual pilus has been developed. Three elongation regions of pili were identified. In region I, P pili stretch elastically, up to a relative elongation of 16 +/- 3%. The product of elasticity modulus and area of a P pilus, EA, was assessed to 154 +/- 20 pN (n=6). In region II, the quaternary structure of the PapA rod unfolds under a constant force of 27 +/- 2 pN (n approximately 100) by a sequential breaking of the interactions between adjacent layers of PapA subunits. This unfolding can elongate the pilus up to 7 +/- 2 times. In region III, pili elongate in a nonlinear manner as a result of stretching until the bond ruptures.

Bacterial Adhesion↗
PubMed2004

Optical tweezers based force measurement system for quantitating binding interactions: system design and application for the study of bacterial adhesion.

An optical force measurement system for quantitating forces in the pN range between micrometer-sized objects has been developed. The system was based upon optical tweezers in combination with a sensitive position detection system and constructed around an inverted microscope. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omnidirectional mechanical spring in response to an external force. The particle's displacement from the equilibrium position is therefore a direct measure of the exerted force. A weak probe laser beam, focused directly below the trapping focus, was used for position detection of the trapped particle (a polystyrene bead). The bead and the condenser focus the light to a distinct spot in the far field, monitored by a position sensitive detector. Various calibration procedures were implemented in order to provide absolute force measurements. The system has been used to measure the binding forces between Escherichia coli bacterial adhesins and galabiose-functionalized beads.

Bacterial Adhesion↗
PubMed2003

Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin.

The ClyA protein is a pore-forming cytotoxin expressed by Escherichia coli and some other enterobacteria. It confers cytotoxic activity toward mammalian cells, but it has remained unknown how ClyA is surface exposed and exported from bacterial cells. Outer-membrane vesicles (OMVs) released from the bacteria were shown to contain ClyA protein. ClyA formed oligomeric pore assemblies in the OMVs, and the cytotoxic activity toward mammalian cells was considerably higher than that of ClyA protein purified from the bacterial periplasm. The redox status of ClyA correlated with its ability to form the oligomeric pore assemblies. In bacterial cells with a defective periplasmic disulphide oxidoreductase system, the ClyA protein was phenotypically expressed in a constitutive manner. The results define a vesicle-mediated transport mechanism in bacteria, and our findings show that the localization of proteins to OMVs directly may contribute to the activation and delivery of pathogenic effector proteins.

Animals↗
PubMed2003

Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration.

The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 degrees C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca(2+) the protofilaments are present as annular structures with a diameter of 40-50 nm. In the presence of 10 mM CaCl(2) the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, where the protein is more destabilised compared to pH 4.5, fibril formation occurs at 37 degrees C and 57 degrees C. At pH 2.0, both ring-shaped and linear protofilaments are formed, in which periodic repeats of ca 35 nm can be distinguished clearly. The rings constitute about 10% of all fibrillar species under these conditions and they are characterised by a larger diameter of 70-80 nm. All the structures bind Congo red and thioflavine T in a manner similar to fibrils associated with a variety of amyloid diseases. At pH 2.0, fibril formation is accompanied by some acidic hydrolysis, producing specific fragmentation of the protein, leading to the accumulation of two peptides in particular, consisting of residues 1-80 and 54-125. At the initial stages of incubation, however, full-length equine lysozyme represents the dominant species within the fibrils. We propose that the ring-shaped structures observed here, and in the case of disease-associated proteins such as alpha-synuclein, could be a second generic type of amyloid structure in addition to the more common linear fibrils.

Amyloid↗
PubMed2003

Role for the major outer-membrane protein from Vibrio anguillarum in bile resistance and biofilm formation.

Vibrio anguillarum, a fish pathogen, produces a 38 kDa major outer-membrane porin, which may be involved in environmental adaptation. The gene encoding the 38 kDa porin was cloned and deleted. The deduced protein sequence was 75 % identical to that of the major outer-membrane protein (OMP), OmpU, from Vibrio cholerae. LacZ expression from an ompU : : lacZ transcriptional gene fusion was increased 1.5-fold in the presence of bile salts and was decreased 50- to 100-fold in a toxR mutant compared to that in the wild-type, showing that ompU expression is positively regulated by ToxR and induced by bile salts. Similar to a toxR mutant, an ompU mutant showed a slight decrease in motility, an increased sensitivity to bile salts and a thicker biofilm with better surface area coverage compared to that of the wild-type. When ompU was expressed under a ToxR-independent promoter in the toxR mutant, the phenotypes for bile resistance and biofilm formation, but not motility were complemented to that of the wild-type. In rainbow trout, the ompU mutant showed wild-type virulence via immersion into infected seawater and intraperitoneal injection. The ompU mutant produced two colony morphologies: opaque, which did not grow at 0.2 % bile, and translucent, which grew at 2 % bile. The translucent ompU mutant strain produced a second major OMP that was induced by bile. All ompU mutants showed variations in the amount and length of smooth LPS. In V. anguillarum, OmpU is not required for virulence, possibly due to a second OMP also critical for resistance to bile; however, outside of the fish host, OmpU limits the progression of biofilm formation.

Adhesins, Bacterial↗
PubMed2003

Potential uses of probiotics in clinical practice.

Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. There is now mounting evidence that selected probiotic strains can provide health benefits to their human hosts. Numerous clinical trials show that certain strains can improve the outcome of intestinal infections by reducing the duration of diarrhea. Further investigations have shown benefits in reducing the recurrence of urogenital infections in women, while promising studies in cancer and allergies require research into the mechanisms of activity for particular strains and better-designed trials. At present, only a small percentage of physicians either know of probiotics or understand their potential applicability to patient care. Thus, probiotics are not yet part of the clinical arsenal for prevention and treatment of disease or maintenance of health. The establishment of accepted standards and guidelines, proposed by the Food and Agriculture Organization of the United Nations and the World Health Organization, represents a key step in ensuring that reliable products with suitable, informative health claims become available. Based upon the evidence to date, future advances with single- and multiple-strain therapies are on the horizon for the management of a number of debilitating and even fatal conditions.

Adult↗
PubMed2003

Characterization of dominantly negative mutant ClyA cytotoxin proteins in Escherichia coli.

We report studies of the subcellular localization of the ClyA cytotoxic protein and of mutations causing defective translocation to the periplasm in Escherichia coli. The ability of ClyA to translocate to the periplasm was abolished in deletion mutants lacking the last 23 or 11 amino acid residues of the C-terminal region. A naturally occurring ClyA variant lacking four residues (183 to 186) in a hydrophobic subdomain was retained mainly in the cytosolic fraction. These mutant proteins displayed an inhibiting effect on the expression of the hemolytic phenotype of wild-type ClyA. Studies in vitro with purified mutant ClyA proteins revealed that they were defective in formation of pore assemblies and that their activity in hemolysis assays and in single-channel conductance tests was at least 10-fold lower than that of the wild-type ClyA. Tests with combinations of the purified proteins indicated that mutant and wild-type ClyA interacted and that formation of heteromeric assemblies affected the pore-forming activity of the wild-type protein. The observed protein-protein interactions were consistent with, and provided a molecular explanation for, the dominant negative feature of the mutant ClyA variants.

Amino Acid Sequence↗
PubMed2003

Transcriptional analysis of the sfa determinant revealing mmRNA processing events in the biogenesis of S fimbriae in pathogenic Escherichia coli.

Among the virulence factors present in pathogenic extraintestinal Escherichia coli strains, expression of fimbrial adhesins is necessary for attachment to the host tissues and subsequent colonization. Occurrence of the sfa determinant coding for the S fimbriae is widespread among the uropathogens and meningitis isolates. The sfa operon consists of nine genes. In the biogenesis of S fimbriae, the proteins encoded by the sfa genes are presumably required in a specific stoichiometry. In the present work we studied how differential expression of the sfa operon genes occurs. Our findings indicate that a number of endoribonucleolytic cleavages occur in the mRNA from the sfa operon, and we detected the presence of different distinct transcriptional products, including sfaBA, sfaA, sfaADE, and sfaGSH. The sfaGSH transcript represents the three distal genes of the sfa operon, which code for the minor subunits of the S fimbriae. Analysis of the proteins in S fimbriae suggested that expression of the sfaGSH transcript provides equimolar amounts of the minor subunits. Furthermore, we showed that in the generation of the major sfaA transcript, the processing included RNase E endoribonuceolytic cleavage of the precursor sfaBA transcript. We suggest that posttranscriptional mRNA processing events result in differential gene expression important to achieve the stoichiometry necessary for fimbrial adhesin biogenesis.

Adhesins, Escherichia coli↗
PubMed2002

Disulfide-bond formation in the transthyretin mutant Y114C prevents amyloid fibril formation in vivo and in vitro.

The Y114C mutation in human transthyretin (TTR) is associated with a particular form of familial amyloidotic polyneuropathy. We show that vitreous aggregates ex vivo consist of either regular amyloid fibrils or disordered disulfide-linked precipitates that maintain the ability to bind Congo red. Furthermore, we demonstrate in vitro that the ATTR Y114C mutant exists in three forms: one unstable but nativelike tetrameric form, one highly aggregated form in which a network of disulfide bonds is formed, and one fibrillar form. The disulfide-linked aggregates and the fibrillar form of the mutant can be induced by heat induction under nonreduced and reduced conditions, respectively. Both forms are recognized by the amyloid specific antibody MAB(39-44). In a previous study, we have linked exposure of this epitope in TTR to a three-residue shift in beta-strand D. The X-ray crystallographic structure of reduced tetrameric ATTR Y114C shows a structure similar to that of the wild type but with a more buried position of Cys10 and with beta-mercaptoethanol associated with Cys114, verifying the strong tendency for this residue to form disulfide bonds. Combined with the ex vivo data, our in vitro findings suggest that ATTR Y114C can lead to disease either by forming regular unbranched amyloid fibrils or by forming disulfide-linked aggregates that maintain amyloid-like properties but are unable to form regular amyloid fibrils.

Adult↗
PubMed2002

Discovery of potent inhibitors of PapG adhesins from uropathogenic Escherichia coli through synthesis and evaluation of galabiose derivatives.

The synthesis of two galabioside (Galalpha1-4Gal) collections based on diversification at the O-1 and O-3' atoms is reported. The galabiosides were evaluated as inhibitors of hemagglutination of human erythrocytes by two strains of Escherichia coli that expressed the class I and class II PapG adhesins, respectively. The class I adhesin was found to prefer aromatic substituents both at the O-1 and the O-3' position of the galabiose disaccharide. One galabioside, p-methoxyphenyl [3-O-(m-nitrobenzyl)-alpha-D-galactopyranosyl]-(1-4)-beta-D-galactopyranoside], had an IC(50) value of 4.1 microM, which is the best inhibition of the class I adhesin to date. In the case of the class II adhesin, one inhibitor, 2-[(S)-2-methoxycarbonyl-2-acetamido-ethylthio]ethyl (3-O-3-[2-(methoxycarbonylphenylthio)propyl]-alpha-D-galactopyranosyl)-(1-4)-alpha-D-galactopyranoside, was found to have an IC(50) value of 68 microM, which is the best artificial inhibition of the class II adhesin reported so far with an affinity for the adhesin comparable to that of the natural tetrasaccharide ligand globotetraose.

Adhesins, Escherichia coli↗
PubMed2002

VanT, a homologue of Vibrio harveyi LuxR, regulates serine, metalloprotease, pigment, and biofilm production in Vibrio anguillarum.

Vibrio anguillarum possesses at least two N-acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi. In this study, we have cloned an additional gene of this circuit, vanT, encoding a V. harveyi LuxR-like transcriptional regulator. A V. anguillarum Delta vanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ. Three lacZ fusions (serA::lacZ, hpdA-hgdA::lacZ, and sat-vps73::lacZ) were identified which exhibited decreased expression in the Delta vanT strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production. V. anguillarum strains require an active VanT to produce high levels of an L-tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation. A V. anguillarum Delta vanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms. Hence, a new member of the V. harveyi LuxR transcriptional activator family has been characterized in V. anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production.

Biofilms↗
PubMed2002

A ToxR homolog from Vibrio anguillarum serotype O1 regulates its own production, bile resistance, and biofilm formation.

ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V. anguillarum serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The toxR mutant but not the toxS mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V. cholerae, and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.

Animals↗