Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

Comparison of secretion of a hepatitis C virus glycoprotein in Saccharomyces cerevisiae and Kluyveromyces lactis.

A C-terminally truncated form of the hepatitis C virus (HCV) putative envelope glycoprotein E2 was expressed in two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis, using a yeast signal peptide sequence to direct the viral glycoprotein to the endoplasmic reticulum (ER) pathway of secretion. Characterization of secreted E2 showed that the protein is endoglycosidase-H-sensitive in both yeasts. Moreover, in vivo inhibition of glycosylation with tunicamycin prevented secretion of E2 and showed that, of its 11 putative N-linked glycosylation sites, at least eight were core-glycosylated. Analysis of the heterologous glycoprotein by SDS-PAGE under nonreducing conditions and by gel filtration demonstrated the formation of multiple disulphides, which resulted in secretion of heterogeneous aggregates with an average molecular mass of 770-1000 kDa in both yeasts. However, variations were observed in the binding of the glycoprotein secreted by the two yeasts to a mannose-specific lectin, and also in its reactivity with anti-E2-specific antibodies. This denotes differences between the two yeasts in folding and/or modification of the E2 glycoprotein.

Integration of minitransposons for expression of the Escherichia coli elt genes at a preferred site in Salmonella typhimurium identifies a novel putative fimbrial locus.

An asd-complementing mini-Tn5 transposon was constructed for random insertion of the Escherichia coli LT enterotoxin genes (elt) into the genome of Deltaasd attenuated strains of Salmonella typhimurium. Transfer of the minitransposon to different S. typhimurium strains resulted in random integration only in strain chi4072, while in strain chi3987, which harbours the virulence plasmid, over 20% of the insertions occurred at the same site. Expression of elt was found to be highest in Salmonella isolates carrying the mini-Tn5 integrated at the preferred site, which was mapped to an uncharacterised region of the virulence plasmid. Sequence analysis of the integration site showed that it lies within an open reading frame with sequence similarity to E. coli leuO and contiguous to a novel fimbrial locus.

Levels of expression and immunogenicity of attenuated Salmonella enterica serovar typhimurium strains expressing Escherichia coli mutant heat-labile enterotoxin.

The effects of heterologous gene dosage as well as Salmonella typhimurium strain variability on immune response toward both the heterologous antigen, the nontoxic mutant of the Escherichia coli heat-labile enterotoxin LTK63, and the carrier Salmonella strain have been analyzed. Effects of a single integration into the host DNA and different-copy-number episomal vectors were compared in S. typhimurium delta cya delta crp delta asd strains of two different serotypes, UK-1 and SR-11. Expression of the enterotoxin in the different Salmonella isolates in vitro was found to vary considerably and, for the episomal vectors, to correlate with the plasmid copy number. LTK63-specific serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) antibodies were highest in mice immunized with the high-level-expression strain. High anti-LTK63 IgG and IgA titers were found to correspond to higher anti-Salmonella immunity, suggesting that LTK63 exerts an adjuvant effect on response to the carrier. Statistically significant differences in anti-LTK63 immune response were observed between groups of mice immunized with the attenuated delta cya delta crp UK-1 and SR-11 derivatives producing the antigen at the same rate. These data indicate that the same attenuation in S. typhimurium strains of different genetic backgrounds can influence significantly the immune response toward the heterologous antigen. Moreover, delivery of the LTK63 enterotoxin to the immune system by attenuated S. typhimurium strains is effective only when synthesis of the antigen is very high during the initial phase of invasion, while persistence of the S. typhimurium strain in deep tissues has only marginal influence.

Gastric antisecretory and anti-ulcer actions of IL-1 in rat involve different IL-1 receptor types.

Limited knowledge exists concerning the interleukin-1 (IL-1) receptor type (IL-1RT) mediating the potent antisecretory and gastro-protective actions of IL-1. In the present study, the gastric actions of IL-1 beta and two related mutant proteins, yIL-1 beta delta 4, an analogue that preferentially binds to IL-1-RTII, and mutant yIL-1 beta N7/Q, an analogue that has equal affinity as IL-1 beta for IL-1RTI and IL-1RTII, have been compared. Modulation of IL-1 gastric actions were also investigated using monoclonal antibody (MAb) preparations raised against IL-1RTI or IL-1RTII. In the pylorus-ligated rat, yIL-1 beta delta 4, yIL-1 beta N7/Q, and IL-1 beta (all at 1 microgram/kg ip) reduced gastric acid secretion (50, 79, and 78%, respectively), indicating the importance of IL-1RTII binding for antisecretory activity. This was further substantiated in experiments using the MAb preparations, which showed that IL-1 beta (1 microgram/kg ip) antisecretory activity was reversed by MAb IL-1RTII (10-50 micrograms/kg sc) but not by MAb IL-1RTI (50 micrograms/kg sc). In contrast, at dosages 10-fold higher (10 micrograms/kg ip) than that used in the study to inhibit acid secretion, IL-1 beta and yIL-1N7/Q equally reduced (approximately 80%) indomethacin-induced gastric damage, but yIL-1 beta delta 4 was ineffective. The results using yIL-1 beta delta 4 indicated that impairment of IL-1RTI binding capacity appeared to be paralleled by a decreased gastroprotective effect.(ABSTRACT TRUNCATED AT 250 WORDS)

A kluyveromyces lactis gene homologue to AAC2 complements the Saccaromyces cerevisiae op1 mutation.

A mutation (op1) in the Saccharomyces cerevisiae AAC2 gene, which codes for the most abundant ADP/ATP carrier isoform, results in lack of mitochondrial-dependent growth and in an as yet unexplained petite-negative phenotype. A gene from the petite-negative yeast Kluyveromyces lactis has been isolated by complementing in multicopy the op1 mutation of S. cerevisiae. This gene, designated KIAAC, can complement the petite-negative phenotype of op1 as well as its inability to grow on nonfermentable carbon sources. KIAAC contains a 915-base pair open reading frame coding for a protein of 305 amino acids which shows a high degree of identity to AAC2. The K. lactis ADP/ATP carrier also shares identity with other known ADP/ATP carrier sequences. In particular, the degree of identity of KIAAC is higher with the Neurospora crassa carrier (80.1%) than with AAC1 (76.6%). The nucleotide sequence upstream of the KIAAC coding region was found to contain a long DNA segment with no coding potential, but presenting features of highly regulated promoter sequences.

Conceivable difference in the anti-inflammatory mechanisms of lipocortins 1 and 5.

Human recombinant lipocortins (LCT) 1 and 5 have been expressed in a yeast secretion vector and purified by ion exchange chromatography. The action of the proteins has been investigated in two models of experimental acute inflammation in the rat: carrageenin induced paw oedema and zymosan induced pleurisy. The effects of the proteins on PGE(2) release in vitro by rat macrophages stimulated with zymosan and on rat neutrophil chemotaxis induced by FMLP have also been assessed. LCT-1 significantly inhibited both paw swelling in carrageenin oedema and leukocyte migration in zymosan pleurisy. Moreover it showed a dose dependent, inhibitory effect on PGE(2) release. Neutrophil chemotaxis was only weakly affected by LCT-1. Conversely LCT-5 did not reduce carrageenin oedema and slightly inhibited PGE(2) release, but showed profound, dose dependent inhibitory activity on leukocyte migration in zymosan pleurisy and on neutrophil chemotaxis. These data suggest that LCT-1 acts mainly by interfering with arachidonic acid metabolism via the inhibition of phospholipase A(2). The anti-inflammatory activity of LCT-5, at variance with LCT-1, may be due to a direct effect on cell motility in addition to the interference with arachidonic acid metabolism.

Expression of cytochrome P450 in yeast after different chemical treatments.

In Saccharomyces cerevisiae a number of chemical agents induce synthesis of cytochrome P450. A cytochrome P450 gene has been well characterized in this yeast: CYP51, which codes for a constitutive enzyme involved in the 14 alpha-demethylation of lanosterol, a key step in the biosynthesis of ergosterol. In this work, we have analysed the level of transcription of the CYP51 gene in correlation with cytochrome P450 enzymatic activity after treatment with several chemical agents known to interact with cytochrome P450. Using as a probe a DNA fragment whose identity to the CYP51 gene was established by sequence analysis and mapping on chromosome VIII, a unique RNA species was observed in all treatment samples. The increased level found for this transcript in cells treated with ethanol, 20% glucose, phenobarbital or 5-methoxypsoralen correlates with the levels of induction in cytochrome P450 enzymatic activity measured in cells grown under the same conditions, indicating that induction of cytochrome P450 by these treatments is regulated at the transcriptional level.

Kluyveromyces lactis rDNA as a target for multiple integration by homologous recombination.

Gene targeting to a single chromosomal locus has been extensively used in Saccharomyces cerevisiae. In this study, we have analyzed targeting of a repetitive sequence, the 25S rDNA gene, to the chromosomal rDNA cluster of Kluyveromyces lactis by the use of a replacement vector. We have obtained K. lactis transformants carrying multiple copies of the replacement cassette inserted into the rDNA chromosomal locus. Analysis of several transformants has shown that the number of integrated copies could range from 4 to 40. Moreover, the distribution of integration sites within the rDNA locus was found to differ in most transformants. Single-copy integration at multiple sites, rather than multicopy integration at a very limited number of sites, was found to be the most frequent event. Also, in most transformants, integration sites were distributed at random as well as in an orderly fashion, i.e., in contiguous or alternate rDNA repeats, suggesting that amplification of the integrated sequences, rather than multiple integration events, may account for the copy number of insertions.

Deletions and rearrangements in Kluyveromyces lactis mitochondrial DNA.

Three classes of respiratory deficient mutants have been isolated from a fusant between Kluyveromyces lactis and Saccharomyces cerevisiae that contains only K. lactis mtDNA. One class (15 isolates), resemble rho 0 mutants of S. cerevisiae as they lack detectable mtDNA. A second class (16 isolates), resemble point mutations (mit-) or nuclear lesions (pet-) of S. cerevisiae as no detectable change is found in their mtDNA. The third class (five isolates), with deletions and rearrangements in their mtDNA are comparable to S. cerevisiae petite (rho-) mutants. Surprisingly, three of the five deletion mutants have lost the same 8.0 kb sector of the mtDNA that encompasses the entire cytochrome oxidase subunit 2 gene and the majority of the adjacent cytochrome oxidase subunit 1 gene. In the other strains, deletions are accompanied by complex rearrangements together with substoiciometric bands and in one instance an amplified sector of 800 bp. By contrast to G + C rich short direct repeats forming deletion sites in S. cerevisiae mtDNA, excision of the 8.0 kb sector in K. lactis mtDNA occurs at an 11 bp A + T rich direct repeat CTAATATATAT. The recovery of three strains manifesting this deletion suggests there are limited sites for intramolecular recombination leading to excision in K. lactis mtDNA.

Nitrite reductase from Pseudomonas aeruginosa: sequence of the gene and the protein.

The gene coding for nitrite reductase of Pseudomonas aeruginosa has been cloned and its sequence determined. The coding region is 1707 bp long and contains information for a polypeptide chain of 568 amino acids. The sequence of the mature protein has been confirmed independently by extensive amino acid sequencing. The amino-terminus of the mature protein is located at Lys-26; the preceding 25 residue long extension shows the features typical of signal peptides. Therefore the enzyme is probably secreted into the periplasmic space. The mature protein is made of 543 amino acid residues and has a molecular mass of 60,204 Da. The c-heme-binding domain, which contains the only two Cys of the molecule, is located at the amino-terminal region. Analysis of the protein sequence in terms of hydrophobicity profile gives results consistent with the fact that the enzyme is fully water soluble and not membrane bound; the most hydrophilic region appears to correspond to the c-heme domain. Secondary structure predictions are in general agreement with previous analysis of circular dichroic data.

Use of ZetaPrep cartridge for the purification of human recombinant interleukin 1 beta.

Zetaprep mass ion-exchange media represent a rapid and efficient chromatographic tool in the separation of proteins, in place of the conventional agarose or cellulose-based gels. We adopted this method, combined with classical steps, to purify to homogeneity human recombinant interleukin 1 beta (IL-1 beta) produced from E. coli and from S. cerevisiae. An anion exchanger QAE-ZetaPrep was used to achieve a rapid partial purification of both proteins. The IL-1 beta purification was completed by gel permeation chromatography on Sephadex G-50. When the protein was produced from yeast, an intermediate chromatographic step on a hydroxylapatite column was also necessary. The isolated proteins proved to be homogeneous by electrophoresis and amino acid analysis. The biological activity of IL-1 beta produced by E. coli is comparable to that of the natural protein, while the protein produced by yeast showed very low specific activity.

Expression of diphtheria toxin fragment A and hormone-toxin fusion proteins in toxin-resistant yeast mutants.

Mutants of the eukaryote Saccharomyces cerevisiae, previously selected for resistance to diphtheria toxin, were investigated for their suitability as hosts for the expression of tox-related proteins. The structural gene for the toxin, encoding the fragment A catalytic domain, was modified for efficient intracellular expression in eukaryotes and placed downstream of the yeast GAL1 promoter element in a plasmid. Transformed mutant yeast grown in galactose, which induces that promoter, were viable and contained active fragment A. In contrast, sensitive, wild-type cells harboring this plasmid grew normally under repressing conditions but were killed when the GAL1 promoter was induced. Additional constructions were also prepared that included sequences encoding either the lymphocyte growth factor interleukin 2 or alpha-melanocyte-stimulating hormone along with the lipid-associating domains of fragment B and the leader peptide of the Kluyveromyces lactis killer toxin. Resistant mutant strains transformed with these plasmids efficiently expressed and secreted the expected chimeric toxins.

Genome organization of the killer plasmid pGK12 from Kluyveromyces lactis.

We have determined the entire sequence of the plasmid K2 from Kluyveromyces lactis which is involved in the maintenance of both killer plasmids in the cell. K2 shares many of the characteristics of the smaller killer plasmid K1: high A+T content (74.7%) and very compact genomic organization. K2 contains ten open reading frames. Some of them overlap on different strands and some on the same strand. Northern blotting of K2 transcripts shows that at least eight ORFs are transcribed. Analysis of the predicted aminoacid sequence of ORF2 from K2 reveals homology with the aminoacid sequence of ORF 1 from K1 and with several viral DNA polymerases. The sequence of K2 from Saccaromyces cerevisiae F102-2 was also determined. Only one nucleotide difference was found between the K2 sequence from the two yeasts. This mutation does not change the genome organization of the plasmid and has only minimal effect on the structure of the encoded proteins.

A novel leader peptide which allows efficient secretion of a fragment of human interleukin 1 beta in Saccharomyces cerevisiae.

Killer strains of Kluyveromyces lactis secrete a toxin which presumably is processed during secretion from a larger precursor. Analysis of the sequence of the K. lactis killer toxin gene predicts that the first 16 amino acids at the amino terminus of the protein should represent its leader peptide. We have tested the capability of this leader peptide to direct secretion of a protein fused to it by inserting a synthetic oligonucleotide identical to the sequence of the putative leader peptide into a yeast expression vector. Subsequently, the cDNA coding for the secreted active portion of the human interleukin 1 beta (IL-1 beta) was fused to the leader peptide sequence of the killer toxin. This construction in Saccharomyces cerevisiae is capable of directing synthesis and secretion of correctly processed IL-1 beta into the culture medium.

An unexpected response to Torulopsis glabrata fusion products to X-irradiation.

Intra-species fusion products of Saccharomyces cerevisiae, Saccharomyces unisporus and Torulopsis glabrata have been isolated following polyethylene glycol-induced fusion of protoplasts and selection for prototrophic colonies. Staining with lomofungin showed that all fusion products were uninucleate. Measurement of DNA content mostly gave values between haploid and diploid levels indicating that the majority of fusion products were aneuploid, Nevertheless fusion products of S. cerevisiae and S. unisporus were, as expected, more resistant to X-irradiation than their haploid parents. By contrast, the X-ray dose-response curve of all T. glabrata fusion products was indistinguishable from their progenitors despite the fact that mitotic segregants could be recovered amongst the survivors to X-rays. A possible explanation for the behaviour towards X-rays of T. glabrata fusion products is that this species lacks a DNA repair pathway involving recombination between homologous chromosomes. We conclude from this study that the shape of the X-ray dose-response curve should not be taken to indicate the ploidy of new yeast isolates without supporting data.