Comprehensive detection of immunorelevant Borrelia garinii antigens by two-dimensional electrophoresis.

Lyme disease is caused by spirochetes of the Borrelia burgdofferi complex and has been reported in many temperate parts of the Northern Hemisphere. The B. burgdorferi complex consists of at least five different species and five genotypes with different pathogenicity. Serodiagnosis was achieved by detection of antigens on one-dimensional (1-D) immunoblots. A systematic and comprehensive approach to elucidate antigens has been started here by the combination of two-dimensional electrophoresis (2-DE) immunoblotting with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Antigens in the proteome of B. garinii BITS were analyzed for their reactivity with sera from patients in early stage (erythema migrans) and late manifestations (neuroborreliosis late, arthritis and acrodermatitis chronica athrophicans) of borreliosis. A strategy to handle the enormous amount of data was developed and 65 antigens were detected, of which 20 were identified. These comprise the known antigens from 1-D immunoblots used routinely in serodiagnosis and additionally the two new antigens, GAPDH and the ABC transporter oligopeptide permease. Several disease-stage unique proteins were detected and some of them identified. The genetic variability between B. garinii strains BITS and 20047, B. afzelii, and B. burgdofferi, sensu stricto, seen on the 2-DE patterns underlines the necessity of the search for additional antigens to improve the serodiagnosis and development of vaccines to be used outside of Northern America. A 2-DE database of B. garinii was built up and is available on the World Wide Web (http://www.mpiib-berlin.mpg.de/2D-PAGE).

Immune response to natural and recombinant antigens of Helicobacter pylori in patients with dyspeptic complaints.

Sera of 223 dyspeptic patients with endoscopic findings of nonulcer dyspepsia (72%), gastric ulcer (15%) and duodenal ulcer (13%) were tested for antibodies against Helicobacter pylori with an enzyme immunoassay and an immunoblot technique using lysates of Helicobacter pylori cells as antigen source. One hundred and fifty-one (68%) sera were found to be positive for Helicobacter pylori IgG with both methods; 5% of the positive results in the enzyme immunoassay were false-positive due to cross-reactions mainly of proteins with a molecular mass of 43-66 kDa. Since cross-reactivity not only reduces the diagnostic value of the immunoassay but also complicates evaluation of the immunoblot results, an attempt was made to overcome these problems by using specific purified recombinant proteins instead of the crude cell preparations as antigens. Of the commonly recognised immunogens of Helicobacter pylori, antibodies against a cell surface protein of 26 kDa, the small urease subunit (29 kDa) and the cytotoxin-associated protein (130 kDa) were identified as highly sensitive serological markers for inclusion in a recombinant antigen mixture for Helicobacter pylori screening. Only the cytotoxin-associated protein was confirmed to be an indicator immunogen for ulcerogenic strains. To assess the reliability of recombinant fragments of this protein in serological screening, the reactivity of antibody to purified fragments of the cytotoxin-associated protein was compared with that to the natural protein. A C-terminal recombinant fragment of 58 kDa showed results identical to those obtained with the natural protein and was thus considered to be an appropriate component of an antigen mixture for serological detection of Helicobacter pylori.

Proteomics in human disease: cancer, heart and infectious diseases.

In recent years, genomics has increased the understanding of many diseases. Proteomics is a rapidly growing research area that encompasses both genetic and environmental factors. The protein composition represents the functional status of a biological compartment. The five approaches presented here resulted in the detection of disease-associated proteins. Calgranulin B was upregulated in colorectal cancer, and hepatoma-derived aldose reductase-like protein was reexpressed in a rat model during hepatocarcinogenesis. In these two investigations, attention was focused on one protein, obviously differing in amount, directly after two-dimensional electrophoresis (2-DE). Additional methods, such as enzyme activity measurements and immunohistochemistry, confirmed the disease association of the two candidates resulting from 2-DE subtractive analysis. The following three investigations take advantage of the holistic potential of the 2-DE approach. The comparison of 2-DE patterns from dilated cardiomyopathy patients with those of controls revealed 25 statistically significant intensity differences, from which 12 were identified by amino acid analysis, Edman degradation or matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). A human myocardial 2-DE database was constructed, containing 3300 protein spots and 150 identified protein species. The number of identified proteins was limited by the capacity of our group, rather than by the principle of feasibility. Another field where proteomics proves to be a valuable tool in identifying proteins of importance for diagnosis is proteome analysis of pathogenic microorganisms such as Borrelia burgdorferi (Lyme disease) and Toxoplasma gondii (toxoplasmosis). Sera from patients with early or late symptoms of Lyme borreliosis contained antibodies of various classes against about 80 antigens each, containing the already described antigens OspA, B and C, flagellin, p83/100, and p39. Similarly, antibody reactivity to seven different marker antigens of T. gondii allowed differentiation between acute and latent toxoplasmosis, an important diagnostic tool in both pregnancy and immunosuppressed patients.

Expression of atrC - encoding a novel member of the ATP binding cassette transporter family in Aspergillus nidulans - is sensitive to cycloheximide.

A new member of the ABC superfamily of transmembrane proteins in Aspergillus nidulans has been cloned and characterized. The topology of conserved motifs subgroups AtrC in the P-glycoprotein cluster of ABC permeases, the members of this subfamily, are known to participate in multidrug resistance (MDR) in diverse organisms. Alignment results display significant amino acid similarity to AfuMDR1 and AflMDR1 from Aspergillus fumigatus and flavus, respectively. Northern analysis reveals that atrC mRNA levels are 10-fold increased in response to cycloheximide. Evidence for the existence of eight additional hitherto unpublished ABC transporter proteins in A. nidulans is provided.

The Aspergillus nidulans GATA factor SREA is involved in regulation of siderophore biosynthesis and control of iron uptake.

A gene encoding a new GATA factor from Aspergillus nidulans, sreA, was isolated and characterized. SREA displays homology to two fungal regulators of siderophore biosynthesis: about 30% overall identity to SRE from Neurospora crassa and about 50% identity to URBS1 from Ustilago maydis over a stretch of 200 amino acid residues containing two GATA-type zinc finger motifs and a cysteine-rich region. This putative DNA binding domain, expressed as a fusion protein in Escherichia coli, specifically binds to GATA sequence motifs. Deletion of sreA results in derepression of L-ornithine-N5-oxygenase activity and consequently in derepression of the biosynthesis of the hydroxamate siderophore N,N',N"-triacetyl fusarinine under sufficient iron supply in A. nidulans. Transcription of sreA is confined to high iron conditions, underscoring the function of SREA as a repressor of siderophore biosynthesis under sufficient iron supply. Nevertheless, overexpression of sreA does not result in repression of siderophore synthesis under low iron conditions, suggesting additional mechanisms involved in this regulatory circuit. Consistent with increased sensitivity to the iron-activated antibiotics phleomycin and streptonigrin, the sreA deletion mutant displays increased accumulation of 59Fe. These results demonstrate that SREA plays a central role in iron uptake in addition to siderophore biosynthesis.

Overexpression of nreB, a new GATA factor-encoding gene of Penicillium chrysogenum, leads to repression of the nitrate assimilatory gene cluster.

To investigate the mechanism of nitrogen metabolite repression in the biotechnologically important fungus Penicillium chrysogenum a polymerase chain reaction approach was employed to identify transcription factors involved in this regulatory circuit, leading to the isolation of a new gene (nreB) encoding a 298 amino acid protein. Despite a low overall amino acid sequence identity of approximately 30%, it shares several features with Dal80p/Uga43p and Gzf3p/Nil2p, both repressors in nitrogen metabolism in Saccharomyces cerevisiae. All three proteins contain an N-terminal GATA-type zinc finger motif, displaying 86% amino acid sequence identity, and a putative leucine zipper motif in the C terminus. Northern blot analysis revealed the presence of two nreB transcripts, 1.8 and 1.5 kilobases in length, that differ in polyadenylation sites. The steady state level of both transcripts is subject to nitrogen metabolite repression. The putative DNA binding domain of NREB, expressed as a fusion protein in Escherichia coli, binds in vitro to GATA sites of its own 5'-upstream region as well as in the promoter of the nitrate assimilation gene cluster. Consistent with a role in the regulation of nitrogen metabolism, overexpression of nreB leads to repression of nitrate assimilatory genes. Hence, the simple view of nitrogen regulation by four GATA factors in yeast, but only one key regulator in filamentous ascomycetes seems no longer valid.

Regulated system for heterologous gene expression in Penicillium chrysogenum.

A system for regulated heterologous gene expression in the filamentous fungus Penicillium chrysogenum was established. This is the first heterologous expression system to be developed for this organism. Expression of a recombinant fungal xylanase gene (xylp) and the cDNA for the human tear lipocalin (LCNI) was achieved by placing the encoding sequences under the control of the repressible acid phosphatase gene (phoA) promoter of P. chrysogenum. Secreted recombinant proteins were detected in the growth media of transformed P. chrysogenum cells by means of bioassays, zymogramography, and Western blotting. Levels of transcription and amounts of recombinant proteins secreted varied among transformants, mainly due to the copy number and the integration site of the expression vector on the fungal chromosome.

Molecular analysis of a Penicillium chrysogenum GATA factor encoding gene (sreP) exhibiting significant homology to the Ustilago maydis urbs1 gene.

Employing a PCR-aided strategy, a Penicillium chrysogenum gene (sreP) encoding a putative GATA-transcription factor has been cloned and characterized. Comparison of the genomic and cDNA sequences revealed the presence of an open reading frame (ORF) encoding a protein of 532 amino acids (aa) which is interrupted by two introns. The deduced aa sequence of sreP reveals 50% identity to a regulator of siderophore biosynthesis (URBS1) from Ustilago maydis over a stretch of 200 aa containing two GATA-type zinc finger motifs and a Cys-rich intervening sequence. Northern blot analysis indicated two transcripts of 2.2 and 2.7 kb in approximately equivalent amount. due to two major transcription start sites.

Sequence analysis and expression of the Penicillium chrysogenum nitrate reductase encoding gene (niaD).

The nitrate reductase gene (niaD) of the filamentous fungus Penicillium chrysogenum encodes a protein of 864 amino acids. The derived protein sequence shows 78% and 72% sequence identity to the corresponding Aspergillus niger and A. nidulans proteins, respectively. The coding region of the Penicillium gene is interrupted by six small introns, as deduced by comparison with the niaD sequences of A. niger and A. nidulans, whereby the positions of the introns are perfectly conserved between these three fungal genes. Northern blot analysis indicated a 2.8 kb transcript and showed that expression of this gene is controlled at the level of mRNA accumulation depending on both induction by nitrate and nitrogen metabolite derepression. Induction of transcription of niaD was found to be paralleled by expression of the major nitrogen regulatory gene nre.

Cloning, structural organization and regulation of expression of the Penicillium chrysogenum paf gene encoding an abundantly secreted protein with antifungal activity.

An abundantly secreted, highly basic 12-kDa protein (PAF) was purified from the culture medium of Penicillium chrysogenum (Pc). Based on the N-terminal amino acid (aa) sequence of the protein, an oligodeoxyribonucleotide probe was derived and used for amplification of the encoding cDNA by PCR. This cDNA fragment encodes a Cys-rich preproprotein of 92 aa which appears to be processed to a mature product of 55 aa. The deduced aa sequence of the preproprotein reveals 42.6% identity to an antifungal protein (AFP) of Aspergillus giganteus. Agar diffusion tests confirmed that the Pc protein exhibits antifungal activity. In order to investigate the promoter region and the structural organization of the paf gene, a genomic 6-kb fragment was isolated and partially sequenced. Comparison of the nucleotide sequence of the genomic fragment and the cDNA clone revealed the presence of a coding region of 279 bp which is interrupted by two introns of 76 and 68 bp in length. In the promoter region, a typical TATA box, a motif resembling the fungal carbon catabolite repression element, as well as several putative GATA factor binding motifs, were found. Northern blot analysis indicated that the regulation of paf expression occurs at the level of mRNA transcription and is under control of carbon catabolite and nitrogen metabolite repression regulatory circuits.

Cre1, the carbon catabolite repressor protein from Trichoderma reesei.

In order to investigate the mechanism of carbon catabolite repression in the industrially important fungus Trichoderma reesei, degenerated PCR-primers were designed to amplify a 0.7-bp fragment of the cre1 gene, which was used to clone the entire gene. It encodes a 402-amino acid protein with a calculated M(r) of 43.6 kDa. Its aa-sequence shows 55.6% and 54.7% overall similarity to the corresponding genes of Aspergillus nidulans and A. niger, respectively. Similarity was restricted to the aa-region containing the C2H2 zinc finger and several aa-regions rich in proline and basic amino acids, which may be involved in the interaction with other proteins. Another aa-region rich in the SPXX-motif that has been considered analogous to a region of yeast RGR1p, was instead identified as a domain occurring in several eucaryotic transcription factors. The presence of the cre1 translation product was demonstrated with polyclonal antibodies against Cre1, which identified a protein of 43 (+/- 2) kDa in cell-free extracts from T. reesei. A Cre1 protein fragment from the two zinc fingers to the region similar to the aa-sequence of eucaryotic transcription factors, was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. EMSA and in vitro footprinting revealed binding of the fusion protein to the sequence 5'-GCGGAG-3', which matches well with the A. nidulans consensus sequence for CreA binding (5'-SYGGRG-3'). Cell-free extracts of T. reesei formed different complexes with DNA-fragments carrying this binding sites, and the presence of Cre1 and additional proteins in these complexes was demonstrated. We conclude that T. reesei Cre1 is the functional homologue of Aspergillus CreA and that it binds to its target sequence probably as a protein complex.

Sequence and structure of Penicillium chrysogenum phoG, homologous to an acid phosphatase-encoding gene of Aspergillus nidulans.

A Penicillium chrysogenum (Pc) gene (phoG), homologous to an Aspergillus nidulans (An) gene which confers phosphate-non-repressible acid phosphatase (APase) activity, has been cloned and sequenced. The 2.9-kb genomic sequence corresponds to two ORFs of 149 and 1630 bp encoding a protein of 593 amino acids (aa). As verified by cDNA sequencing, the coding region is interrupted by an 85-bp intron. The deduced aa sequence of phoG reveals 61% aa identity to the translated long ORF of the An APase-encoding gene. Northern blot analysis indicated a 2.3-kb transcript in approximately equivalent amount in mycelia grown under different phosphate concentrations.

Amino acid sequence heterogeneity of the chromosomal encoded Borrelia burgdorferi sensu lato major antigen P100.

The entire nucleotide sequence of the chromosomal encoded major antigen p100 of the European Borrelia garinii isolate B29 was determined and the deduced amino acid sequence was compared to the homologous antigen p83 of the North American Borrelia burgdorferi sensu stricto strain B31 and the p100 of the European Borrelia afzelii (group VS461) strain PKo. p100 of strain B29 shows 87% amino acid sequence identity to strain B31 and 79.2% to strain PKo, p100 of strain B31 and PKo shows 62.5% identity to each other. In addition, partial nucleotide sequences of the most heterogeneous region of the p100 gene of two other Borrelia garinii isolates (PBi and VS286) have been determined and the deduced amino acid sequences were compared with all p100 of Borrelia garinii published so far. We found an amino acid sequence identity between 88.6 and 100% within the same genospecies. The N-terminal part of the p100 proteins is highly conserved whereas a striking heterogeneous region within the C-terminal part of the proteins was observed.

Molecular cloning and analysis of nre, the major nitrogen regulatory gene of Penicillium chrysogenum.

We have isolated the Penicillium chrysogenum nre gene which is homologous to the major nitrogen regulatory genes areA from Aspergillus nidulans and nit-2 from Neurospora crassa. Overall, nre shows 60% identity to areA and 30% identity to nit-2 at the amino-acid level. The gene encodes a protein of 835 amino-acid residues and contains a single Cys2/Cys2-type zinc finger with an adjacent basic region and a putative acidic activation region. In the DNA-binding domain, 98% of the amino-acid residues are identical in nre, areA and nit-2. The nre gene has been shown to be functional in N. crassa by heterologous complementation of a nit-2 mutant. Growth tests indicated that transformants could utilize nitrate, amino-acids, purines and amides as sole nitrogen sources. Nitrate reductase activity assays performed with transformants demonstrated that nitrogen control was completely normal. Complementation of N. crassa nit-2 mutants with 5'-deletion clones of nre suggests the possible presence of an internal promoter within the coding region. Northern analysis and ribonuclease protection assays of total cellular RNA indicated that nre encodes a 3.2-kb transcript which is reduced in content under conditions of nitrogen repression.

[The significance of infection pathways for hepatitis C virus associated chronic liver disease]. / Die Bedeutung der Infektionswege für die Hepatitis-C-Virus-assoziierte chronische Lebererkrankung.

The importance of hepatitis C virus (HCV) infection as a cause of chronic liver disease has become clear with the introduction of serologic detection methods. On the basis of epidemiologic evidence the parenteral way of infection has been considered to be the most important one. However, the epidemiologic data regarding the significant route of infection are still limited. To study the ways of HCV-infection and their possible influence on the course of the disease, 73 patients with chronic hepatitis C infection were examined. Setting was the out-patient department of Gastroenterology of our University Hospital. Patients history, completed by a questionnaire, laboratory findings and liver histology were analysed. The study indicated that in 50% of the patients transmission had occurred through parenteral infection, the other 50% had been infected through the non-parenteral (sporadic) way. The study revealed further that the way of infection has an influence on the progression of liver disease with the patients infected sporadically showing histologically more serious hepatic changes. In 50 patients HCV-infection was the only cause of their chronic liver disease, in 23 patients additional pathogenic factors were detected. These 23 patients showed a rapid progress of the disease. Therefore, HCV-infection cannot be considered any longer as a disease that is primarily transmitted parenterally. Due to the large number of sporadic infections, HCV-infection will continue to be of great epidemiologic importance even after the effective elimination of contaminated blood products.

Spontaneous bacterial peritonitis is associated with high levels of interleukin-6 and its secondary mediators in ascitic fluid.

We investigated 37 patients with ascites and liver cirrhosis in order to examine whether on the basis of correlation of cytokines and acute phase proteins of the ascitic fluid, prognosis of spontaneous bacterial peritonitis can be made. Significantly enhanced levels of interleukin-6, as well as acute phase reactants alpha-1-antitrypsin and C-reactive protein were found in the ascitic fluid of patients with spontaneous bacterial peritonitis. The levels of tumour necrosis factor alpha (TNF-alpha), neopterin, interleukin 2-receptor and granulocyte-macrophage colony stimulating factor were higher in patients with spontaneous bacterial peritonitis, but without statistical significance, whereas no differences were found between the interferon gamma, interleukin-2 and interleukin-1 levels. In addition, interleukin-6, TNF-alpha and neopterin levels were found to correlate significantly with the outcome of the disease. These findings show that acute phase reaction occurs in the ascitic compartment in correlation with the development of spontaneous bacterial peritonitis.

Isolation and analysis of a linear plasmid-located gene of Borrelia burgdorferi B29 encoding a 27 kDa surface lipoprotein (P27) and its overexpression in Escherichia coli.

Using an antiserum of a patient with cutaneous manifestations of Lyme borreliosis we have isolated the gene encoding a 27 kDa protein antigen (P27) of Borrelia burgdorferi B29 from a lambda-gt11 expression library. Nucleotide sequence analysis revealed that it is a basic protein of 248 amino acids with a typical prokaryotic leader sequence of 17 amino acid residues at the N-terminus of the proposed translation product. Biochemical investigations showed that P27 is a surface-exposed lipoprotein. From pulsed-field gel electrophoresis and subsequent Southern blot analysis it is evident that the p27 gene is located on a linear plasmid of a size of approximately 55 kb. It was overexpressed in Escherichia coli and the purified recombinant protein was used for biochemical and serological studies. Northern and Western blot analysis demonstrated that p27 is expressed in the European B. burgdorferi strain B29, but not in the American strain B31.

Cloning and structural organization of a xylanase-encoding gene from penicillium chrysogenum.

The filamentous fungus, Penicillium chrysogenum, is able to grow on xylan as a sole carbon source. Under these conditions, high levels of a xylanase (XYLP) are secreted into the medium. After purification and characterization of this enzyme, we have isolated both the encoding cDNA and the genomic sequence by using oligodeoxyribonucleotides derived from partial amino acid (aa) sequences of the purified enzyme. The gene is approximately 1.6 kb in length, and comparison of the nucleotide (nt) sequence of the genomic and the cDNA clone revealed the presence of ten exons and nine introns. All intron/exon splice junctions exactly follow the GT/AG rule, except for the seventh intron which shows atypical AT/AC splice sites. The immediate 5'-flanking region of the first exon contains one putative CCAAT consensus sequence and a perfect TATA box. Primer extension analysis revealed two transcription start points located 38 and 34 nt upstream from the ATG start codon. A sequence of 23 aa representing a typical signal peptide is present at the N terminus of the deduced aa sequence. Northern blot analysis of total cellular RNA indicated that xylP encodes a 1.3-kb transcript which is induced by xylan. The aa sequence of XYLP shows considerable homology to high-M(r) acidic xylanases (Xln) and cellulases from different bacteria, yeasts and fungi.

Effects of adequate versus inadequate treatment of cutaneous manifestations of Lyme borreliosis on the incidence of late complications and late serologic status.

Eighty-two patients who were treated at the Department of Dermatology, Innsbruck, Austria, from 1980 to 1987 for cutaneous manifestations of Lyme disease were subjected to a clinical follow-up investigation aimed at detecting dermatologic, neurologic, and internal late complications of borreliosis. Only 54 of these patients had received adequate antibiotic treatment according to current standards. Also, their sera were investigated for the presence of immunoglobulin G (IgG) and IgM Borrelia burgdorferi antibodies by an indirect immunofluorescence assay, three different enzyme-linked immunosorbent assays, and immunoblotting. As a control, the sera of 126 healthy blood donors were investigated with the same assays. Results showed no unambiguous clinical late complications of Lyme borreliosis, even in inadequately treated or untreated patients. Seropositivity varied considerably according to the assay used; the indirect immunofluorescence assay yielded the highest scores. The proportion of seropositive results (immunofluorescence assay) was 59% in patients with erythema chronicum migrans, 69% in those with lymphocytoma cutis, and 100% in those with acrodermatitis chronica atrophicans (overall 63%); in contrast, only 31% of the blood donor control group were found to be seropositive. Seropositivity did not correlate with adequacy of treatment, interval between onset of symptoms and treatment, time span since treatment, age of patients, and presence of antinuclear antibodies. Immunoblot pattern showed high incidence of antibodies against the 29/31-kD (outer surface proteins OspA and OspB) and 55/58-kD antigens in general and against the 41-kD protein (flagellin) in patients with acrodermatitis chronica atrophicans only.

Purification, characterization and partial amino acid sequences of a xylanase produced by Penicillium chrysogenum.

An extracellular xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8, endo 1,4-beta-xylanase) was found to be the major protein in the culture filtrate of Penicillium chrysogenum when grown on 1% xylan. In contrast to other microorganism no xylanase multiplicity was found in P. chrysogenum under the conditions used. This enzyme was purified to homogeneity by high performance anion-exchange and size-exclusion chromatography. It had an M(r) of 35,000 as estimated by SDS-PAGE and was shown to be active as a monomer. No glycosylation of the protein could be detected neither by a sensitive glycostain nor by enzymatic deglycosylation studies. The enzyme hydrolyzed oat spelt and birchwood xylan randomly, yielding xylose and xylobiose as major end products. It had no cellulase, CMCase, beta-xylosidase or arabinogalactanase activity but acted on p-nitrophenylcellobioside. The pH and temperature optima for its activity were pH 6.0 and 40 degrees C, respectively. Eight peptides obtained after endoproteinase LysC digestion of xylanase have been sequenced, six of them showed considerable amino acid similarity to glucanases and high M(r)/acidic xylanases from different bacteria, yeasts and fungi.