Component-resolved in vitro diagnosis of carrot allergy in three different regions of Europe.

BACKGROUND: Carrot is a frequent cause of food allergy in Europe. The objective of this study was to evaluate a panel of carrot allergens for diagnosis of carrot allergy in Spain, Switzerland and Denmark. METHODS: Forty-nine carrot allergic patients, 71 pollen allergic but carrot-tolerant patients and 63 nonatopic controls were included. Serum IgE to carrot extract, recombinant carrot allergens (rDau c 1.0104; rDau c 1.0201; rDau c 4; the isoflavone reductase-like proteins rDau c IFR 1, rDau c IFR 2; the carrot cyclophilin rDau c Cyc) were analyzed by ImmunoCAP. RESULTS: The sensitivity of the carrot extract-based test was 82%. Use of the recombinant allergens increased the sensitivity to 90%. The Dau c 1 isoforms were major allergens for Swiss and Danish carrot allergic patients, the profilin rDau c 4 for the Spanish patients. The rDau c IFR 1 and rDau c IFR 2 were recognized by 6% and 20% of the carrot allergics, but did not contribute to a further increase of sensitivity. Among pollen allergic controls, 34% had IgE to carrot extract, 18% to each of rDau c 1.0104, rDau c 1.0201 and rDau c 4, 8% to rDau c IFR 1 and 7% to rDau c IFR 2. Sensitization to rDau c Cyc occurred in one carrot allergic patient and one nonatopic control. CONCLUSION: Component-resolved in vitro analyses revealed a significant difference in IgE sensitization pattern between geographical regions and in the prevalence of sensitization to carrot components between carrot allergic and carrot-tolerant but pollen sensitized patients.

Functional domains in the Mig1 repressor.

Mig1 is a zinc finger protein that mediates glucose repression in the yeast Saccharomyces cerevisiae. It is related to the mammalian Krox/Egr, Wilms' tumor, and Sp1 proteins and binds to a GC-rich motif that resembles the GC boxes recognized by these proteins. We have performed deletion mapping in order to identify functional domains in Mig1. We found that a small C-terminal domain comprising the last 24 amino acids mediates Mig1-dependent repression of a reporter gene. This effector domain contains several leucine-proline dipeptide repeats. We further found that inhibition of Mig1 activity in the absence of glucose is mediated by two internal elements in the Mig1 protein. A Mig1-VP16 hybrid activator was used to further investigate how Mig1 is regulated. Mig1-VP16 can activate transcription from promoters containing Mig1-binding sites and suppresses the inability of Snf1-deficient cells to grow on certain carbon sources. We found that a deletion of the SNF1 gene increases the activity of Mig1-VP16 fivefold under derepressing conditions but not in the presence of glucose. This shows that the hybrid activator is under negative control by the Snf1 protein kinase. Deletion mapping within Mig1-VP16 revealed that regulation of its activity by Snf1 is conferred by the same internal elements in the Mig1 sequence that mediate inhibition of Mig1 activity in the absence of glucose.

The MIG1 repressor from Kluyveromyces lactis: cloning, sequencing and functional analysis in Saccharomyces cerevisiae.

Sequence comparisons between Saccharomyces cerevisiae ScMig1 and Aspergillus nidulans CREA proteins allowed us to design two sets of degenerate primers from the conserved zinc finger loops. PCR amplification on Kluyveromyces marxianus and K. lactis genomic DNA yielded single products with sequences closely related to each other and to the corresponding regions of ScMig1 and CREA. The KIMIG1 gene of K. lactis was cloned from a genomic library using the K. marxianus PCR fragment as probe. KIMIG1 encodes a 474-amino acid protein 55% similar to ScMig1. Besides their highly conserved zinc fingers, the two proteins display short conserved motifs of possible significance in glucose repression. Heterologous complementation of a mig1 mutant of S. cerevisiae by the K. lactis gene demonstrates that the function of the Mig1 protein is conserved in these two distantly related yeasts.

Construction and use of a new vector/transposon, pLBT::mini-Tn10:lac:kan, to identify environmentally responsive genes in a marine bacterium.

The previously described pLOFKm transposon delivery plasmid (J.Bacteriol. (1990) 172, 6557-6567) was engineered such that a promotorless lacZ gene was cloned within the transposon cassette, generating the vector pLBT. Using pLBT, stable insertion mutations were generated at high frequencies in Vibrio sp. S141 and Pseudomonas sp. S91, and the interrupted genes could be monitored for their pattern of regulation. Genetic screens isolated mutants defective in a variety of activities. We describe the construction and use of pLBT as a tool for reporter gene mutant analysis in bacteria other than well-characterized laboratory strains.

Enhancement of hazelnut extract for IgE testing by recombinant allergen spiking.

BACKGROUND: Hazelnuts are a common cause of food allergic reactions. Most hazelnut allergic individuals in central and northern Europe are sensitized to Cor a 1, a member of the PR-10 protein family, while the lipid transfer protein Cor a 8 acts as a major allergen in the south of Europe. Other allergens, including profilin and seed storage proteins, may be important in subgroups of patients. Reliable detection of specific IgE in the clinical diagnosis of food allergy requires allergen reagents with a sufficient representation of all relevant allergen components. Some reported observations suggest that natural hazelnut extract may not be fully adequate in this respect. METHODS: The capacity of immobilized natural hazelnut extract to bind Cor a 1-, Cor a 2- and Cor a 8-specific IgE and IgG antibodies was investigated by serum adsorption and extract dilution experiments and by the use of allergen specific rabbit antisera. All measurements were performed with the ImmunoCAP assay platform. RESULTS: The experimental results revealed an incomplete capacity of immobilized hazelnut extract to capture IgE antibodies directed to the major allergen Cor a 1. Spiking of hazelnut extract with recombinant Cor a 1.04 prior to solid phase coupling gave rise to significantly enhanced IgE antibody binding from Cor a 1 reactive sera. The spiking did not negatively affect the measurement of IgE to extract components other than Cor a 1. CONCLUSION: A hazelnut allergen reagent with enhanced IgE detection capacity can be generated by supplementing the natural food extract with recombinant Cor a 1.04.

A critical assessment of allergen component-based in vitro diagnosis in cherry allergy across Europe.

BACKGROUND: Food allergy to cherry occurs throughout Europe, typically with restricted oral reactions in the central and northern parts but with frequent systemic reactions in the Mediterranean region. Previous studies have demonstrated insufficient sensitivity of commercially available cherry extract reagents in the diagnosis of cherry allergy. OBJECTIVE: To assess the diagnostic performance of specific IgE tests based on recombinant cherry allergens in comparison with an extract-based assay and to skin prick test (SPT). A secondary objective was to analyse the frequency of systemic reactions in cherry-allergic subjects across Europe, including the largest population of LTP-sensitized subjects from central Europe studied to date. METHODS: A total of 186 subjects from central Europe and Spain were studied. Serum IgE was analysed with ImmunoCAP tests carrying rPru av 1, 3 and 4, combined and separately, and cherry extract. RESULTS: Among the central European cherry allergics, the mix of rPru av 1, 3 and 4 had a sensitivity of 95%, compared with 65% for cherry extract, and the IgE binding capacity of the recombinant mix was considerably higher. The sensitivity of the two tests was more comparable in the Spanish population, 95% and 86%, respectively. The recombinant allergen ImmunoCAP equalled SPT in terms of sensitivity and specificity. Consistent with previous reports, major geographic differences in sensitization pattern and prevalence of systemic reactions were found. A significantly higher rate of systemic reactions was found in Spanish patients sensitized to Pru av 3 whereas German patients sensitized to LTP only had oral allergy syndrome. CONCLUSIONS: The recombinant cherry allergen ImmunoCAP is a highly sensitive diagnostic tool, clearly superior to any diagnostic method based on cherry extract. Three cherry allergens are sufficient for detecting sensitization in 95% of cherry-allergic subjects. Systemic reactions are common in LTP-sensitized individuals but seem to require at least one additional causative factor.

Negative control of the Mig1p repressor by Snf1p-dependent phosphorylation in the absence of glucose.

Mig1p, a zinc-finger protein that is related to the Krox/Egr, Wilms' tumor and Sp1 proteins, mediates glucose repression in the yeast Saccharomyces cerevisiae. Mig1p is inactive in the absence of glucose, and this inhibition is dependent on the Snf1p (Cat1p) protein kinase. The regulation is mediated by an internal part of Mig1p, and it can be transferred to a Mig1-viral protein 16 (VP16) fusion protein that functions as an activator [Ostling, J., Carlberg, M. & Ronne, H. (1996) Mol. Cell. Biol. 16, 753-761]. We have used Mig1-VP16 to identify three target sites for phosphorylation that mediate Snf1p-dependent inhibition of its activity in the absence of glucose. Two of the sites, Ser278 and Ser311, fit the consensus sequence for phosphorylation by the kinase Snf1p, as determined in vitro. However, a third phosphorylated site, Ser108, does not resemble a Snf1p site. We tested the effect of deleting residues 181-245, which contain two conserved alanine-leucine-serine motifs. We found that the deletion produces a partially constitutive activator, indicating that this region plays a general negative role in regulating Mig1p.

Isolation of a carbon starvation regulatory mutant in a marine Vibrio strain.

A carbon starvation-responding lac fusion of the marine Vibrio sp. strain S14 was used as a reporter strain in order to identify genes critical in the regulation of the carbon starvation response. Interestingly, sequence data together with an altered phenotype with respect to the accumulation of guanosine 3',5'-bispyrophosphate (ppGpp) imply that one of the genes (csrS) identified by this approach is an Escherichia coli spoT equivalent. Complementary data suggest that the function encoded by the csrS gene is essential for the successful development of starvation and stress resistance.

Global analysis of the carbon starvation response of a marine Vibrio species with disruptions in genes homologous to relA and spoT.

The stringent control response, which involves a rapid accumulation of ppGpp, is triggered if the marine Vibrio sp. strain S14 is subjected to carbon and energy starvation. By means of high-resolution two-dimensional gel electrophoresis analysis, we addressed the role of the major ppGpp-synthesizing enzyme (RelA) in the regulation of the carbon starvation response of Vibrio sp. strain S14. The finding that a large number of the carbon starvation-induced proteins were underexpressed in the Vibrio sp. S14 relA mutant strain after the onset of glucose starvation suggests that a rapid accumulation of ppGpp is required for induction of many of the carbon starvation-induced proteins. However, it was also found that a majority of the carbon starvation-induced proteins were significantly less induced if the stringent control response was provoked by amino acid starvation. We therefore also addressed the notion that a carbon starvation-specific signal transduction pathway, complementary to the stringent control, may exist in Vibrio sp. strain S14. It was found that a majority of the proteins that were underexpressed in the relA mutant strain were also underexpressed in the Vibrio sp. S14 spoT mutant strain (csrS1). Interestingly, a large proportion of these underexpressed proteins were found to belong to a group of proteins that are not, or significantly less, induced by starvation conditions that do not promote starvation survival. On the basis of these observations and the finding that the csrS1 strain survives poorly but accumulates ppGpp in a fashion similar to the wild type during carbon and energy source starvation, the gene product of the csrS gene is suggested to be responsible for the mediation of a signal which is complementary to ppGpp and essential for the successful development of the starvation- and stress-resistant cell. This conclusion was also supported by experiments in which changes in phenotypic characteristics known to be induced during carbon starvation were studied. The starvation induction of the high-affinity glucose uptake system was found to be dependent on the csrS gene but not relA, and the synthesis of carbon starvation-specific periplasmic space proteins was dependent, at different times of starvation, on both the relA and the csrS gene products.

Starvation-specific formation of a peripheral exopolysaccharide by a marine Pseudomonas sp., strain S9.

The marine bacterium Pseudomonas sp. strain S9 produces exopolysaccharides (EPS) during both growth and total energy source and nutrient starvation. Transmission electron microscopy of immunogold-labeled cells demonstrated that the EPS is closely associated with the cell surface during growth (integral EPS), while both the integral form and a loosely associated extracellular (peripheral) form were observed during starvation. Formation and release of the latter rendered the starvation medium viscous. In addition, after 3 h of starvation in static conditions, less than 5% of the cells were motile, compared with 100% at the onset of starvation and approximately 80% subsequent to release of the peripheral EPS at 27 h of starvation. Inhibition of protein synthesis with chloramphenicol added before 3 h of starvation caused no increase in viscosity. However, addition of chloramphenicol at 3 h did not prevent the subsequent increase in viscosity displayed by S9 cells. The amount of integral EPS increased for both nontreated and chloramphenicol-treated S9 cells during the first hour of starvation, with a subsequent equal decrease. The chloramphenicol-treated cells, as well as cells of a transposon-generated mutant strain deficient in peripheral EPS formation, remained adhesive to a hydrophobic inanimate surface during the initial 5 h of starvation, whereas nontreated wild-type cells had progressively decreased adhesion capacity. During the initial 5 h of starvation, most of the nontreated cells but only a small fraction of the chloramphenicol-treated and mutant cells detached from the hydrophobic substratum.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative analysis in three fungi reveals structurally and functionally conserved regions in the Mig1 repressor.

The Mig1 repressor is a key effector in glucose repression in the yeast Saccharomyces cerevisiae. To gain further insights into structure-function relationships, we have now cloned the MIG1 homologue from the yeast Kluyveromyces marxianus. The amino acid sequence deduced from KmMIG1 differs significantly from ScMig1p outside the highly conserved zinc fingers. However, 12 discrete conserved motifs could be identified in a multiple alignment that also included the K. lactis Mig1p sequence. We further found that KmMig1p is fully functional when expressed in S. cerevisiae. First, it represses the SUC2 promoter almost as well as ScMig1p. This repression requires the Cyc8 and Tup1 proteins and is dependent on a C-terminal region comprising several conserved leucine-proline repeats. Second, KmMig1p is regulated by glucose in S. cerevisiae, and a KmMig1-VP16 hybrid activator is inhibited by the ScSnf1p kinase in the absence of glucose. This suggests that KmMig1p has retained the ability to interact with several S. cerevisiae proteins, and reinforces the notion that the conserved motifs are functionally important. Finally, we found that the physiological role of Mig1p also is conserved in K. marxianus, since KmMig1p represses INU1, the counterpart of SUC2 in this organism.

Global analysis of physiological responses in marine bacteria.

In this paper, we present results from studies on marine Vibrio species, in which complex adaptive responses have been investigated. The results of two-dimensional polyacrylamide gel electrophoresis serve to illustrate the usefulness of a global approach, and how it can be combined with other methodologies in order to achieve an improved understanding of the means by which bacteria adapt to alterations in environmental conditions. The overall strategies described in this paper are particularly useful for studies of bacteria for which efficient genetic tools, background genotypes and in depth physiological data are not yet available.

Four hydrophobic amino acid residues in the C-terminal effector domain of the yeast Mig1p repressor are important for its in vivo activity.

The Mig1 repressor is a zinc finger protein that mediates glucose repression in yeast. Previous work in Saccharomyces cerevisiae has shown that two domains in Miglp are required for repression: the N-terminal zinc finger region and a C-terminal effector domain. Both domains are also conserved in Miglp homologs from the distantly related yeasts Kluyveromyces lactis and K. marxianus, and these Mig1 proteins can fully replace the endogenous Mig1p in S. cerevisiae. We have now made a detailed analysis of the conserved C-terminal effector domain in Mig1p from K. marxianus, using expression in S. cerevisiae to monitor its function. First, a series of small deletions were made within the effector domain. Second, an alanine scan mutagenesis was carried out across the effector domain. Third, double, triple and quadruple mutants were made that affect certain residues within the effector domain. Our results show that four conserved residues within the effector domain, three leucines and one isoleucine, are particularly important for its function in vivo. The analysis further revealed that while the C-terminal effector domain of KmMig1p mediates a seven- to nine-fold repression of the reporter gene, a five- to sixfold residual effect also exists that is independent of the C-terminal effector domain. Similar results were obtained when the corresponding mutations were made in ScMig1p. Moreover, we found that mutations in these residues affect the interaction between Mig1p and the general corepressor subunit Cyc8p (Ssn6p). Modeling of the C-terminal effector domain using a protein of known structure suggests that it may be folded into an alpha-helix.

Mso1p: a yeast protein that functions in secretion and interacts physically and genetically with Sec1p.

The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane. We previously have cloned two yeast genes encoding syntaxins, SSO1 and SSO2, as suppressors of the temperature-sensitive sec1-1 mutation. We now describe a third suppressor of sec1-1, which we call MSO1. Unlike SSO1 and SSO2, MSO1 is specific for sec1 and does not suppress mutations in any other SEC genes. MSO1 encodes a small hydrophilic protein that is enriched in a microsomal membrane fraction. Cells that lack MSO1 are viable, but they accumulate secretory vesicles in the bud, indicating that the terminal step in secretion is partially impaired. Moreover, loss of MSO1 shows synthetic lethality with mutations in SEC1, SEC2, and SEC4, and other synthetic phenotypes with mutations in several other late-acting SEC genes. We further found that Mso1p interacts with Sec1p both in vitro and in the two-hybrid system. These findings suggest that Mso1p is a component of the secretory vesicle docking complex whose function is closely associated with that of Sec1p.

Extracellular signal molecule(s) involved in the carbon starvation response of marine Vibrio sp. strain S14.

The role of exogenous metabolites as putative signal molecules mediating and/or regulating the carbon starvation adaptation program in Vibrio sp. strain S14 was investigated. Addition of the stationary-phase supernatant extract (SSE) of Vibrio sp. strain S14 to logarithmic-phase cells resulted in a significant number of carbon starvation-induced proteins being up-regulated. Halogenated furanones, putative antagonists of acylated homoserine lactones (AHLs), inhibited the synthesis of proteins specifically induced upon carbon starvation. The effect of the furanone was the opposite of that caused by SSE with respect to the up- and down-regulation of protein expression, indicating that both the furanone and the putative signalling molecules were acting on the same regulatory pathway. Culturability was rapidly lost when Vibrio sp. strain S14 was starved in the presence of the furanone at a low concentration. The furanone also had a negative effect on the ability of carbon-starved cells to mount resistance against UV irradiation and hydrogen peroxide exposure. The SSE of Vibrio sp. strain S14 had the ability to provide cross-protection against the loss in viability caused by the furanone. We have further demonstrated that the SSE taken from low- as well as high-cell-density cultures of Vibrio sp. strain S14 induced luminescence in Vibrio harveyi. Taken together, the results in this report provide evidence that Vibrio sp. strain S14 produces extracellular signalling metabolites during carbon and energy starvation and that these molecules play an important role in the expression of proteins crucial to the development of starvation- and stress-resistant phenotypes.

Two separate regulatory systems participate in control of swarming motility of Serratia liquefaciens MG1.

Swarming motility of Serratia liquefaciens MG1 requires the expression of two genetic loci, flhDC and swrI. Here we demonstrate that the products of the flhDC operon (the flagellar master regulator) and the swrI gene (the extracellular signal molecule N-butanoyl-L-homoserine lactone) are global regulators which control two separate regulons.

How do non-differentiating bacteria adapt to starvation?

Non-differentiating bacteria adapt to starvation induced growth arrest by a complex turn-on/turn-off pattern of protein synthesis. This response shows distinct similarities with those of spore formation in differentiating organisms. A substantial amount of information on the non-growth biology of non-differentiating bacteria can be derived from studies on Vibrio strains. One important result is that carbon rather than nitrogen or phosphorus starvation leads to the development of a starvation and stress resistant cell in these organisms. Hence, we have attempted to characterize the carbon starvation stimulon. By the use of two-dimensional gel electrophoresis of pulse-labelled cells and transposon mutagenesis, using reporter gene constructs, the identity and function of some members of the carbon starvation stimulon have been elucidated. Moreover, regulatory genes of the starvation response have been identified with these techniques. Current studies primarily address the identity and function of these genes. The role of transcript modification and stability for both long term persistence during starvation as well as the efficient recovery of cells which occurs upon nutrient addition is also addressed. It is suggested that an understanding of the functionality of the translational machinery is essential for the understanding of these adaptive pathways. This contribution also discusses the diversity of the differentiation-like response to starvation in different bacteria and whether a general starvation induced programme exists.