Nisin-induced expression of recombinant T cell epitopes of major Japanese cedar pollen allergens in Lactococcus lactis.

Japanese cedar pollinosis is a seasonal allergic disease caused by two major pollen allergens: Cry j 1 and Cry j 2 antigens. To develop an oral vaccine to treat pollinosis, we constructed recombinant Lactococcus lactis harboring the gene encoding fused T cell epitopes from the Cry j 1 and Cry j 2 antigens. The recombinant T cell epitope peptide was designed to contain the fused cholera toxin B subunit as an adjuvant and a FLAG tag at the C-terminus. An expression plasmid was constructed by inserting the T cell epitope peptide gene into the multiple cloning sites of plasmid pNZ8148, an Escherichia coli-L. lactis shuttle vector. The constructed plasmid was transformed into L. lactis NZ9000 for expression induced by nisin, an antibacterial peptide from L. lactis. The expression of the epitope peptide was induced with 10-40 ng/mL nisin, and the expressed T cell epitope peptide was detected by western blot analysis using an anti-FLAG antibody and an antibody against the T cell epitopes. The concentration of the epitope peptide was estimated to be ~ 22 mg/L of culture in the presence of 40 ng/mL nisin, although it varied depending on the nisin concentration, the culture time, and the bacterial concentration when nisin was added. The expression of the recombinant epitope peptide in L. lactis, an organism generally recognized as safe, as demonstrated in this study, may contribute to the development of an oral vaccine for the treatment of pollinosis.

Characterization of LuxI and LuxR Protein Homologs of N-Acylhomoserine Lactone-Dependent Quorum Sensing System in Pseudoalteromonas sp. 520P1.

Pseudoalteromonas sp. 520P1 (hereafter referred to as strain 520P1) produces N-acylhomoserine lactones (AHLs), which serve as signaling molecules in Gram-negative bacterial quorum sensing. In a previous genomic analysis of the 5.25-Mb genome of strain 520P1, we detected the presence of at least one homolog of the AHL synthase gene (luxI) and five homologs of the transcriptional regulator protein gene (luxR). The LuxI homolog of strain 520P1 (PalI) contained the conserved amino acid motifs shared by all the LuxI family proteins of the different species examined here. The palI gene expressed in Escherichia coli produced two types of AHLs. In the thin-layer chromatography analysis, these AHLs showed identical mobility to the AHLs produced by strain 520P1. The five LuxR homologs of strain 520P1 (PalR1-PalR5) shared only 17-34% amino acid sequence identity, although higher identities were observed in the C-terminal DNA-binding domain. Among the five PalRs, only PalR5 displayed close homology with LuxR family proteins from other Pseudoalteromonas strains. Notably, the palR3 and palI genes were located close together and only 1021 bases apart in the genome. No cognate luxI homolog associated with the four other palR genes was detected. These characteristics of PalI and the PalRs suggest that AHL autoinducers generated by the PalI enzyme might regulate cellular metabolism in cooperation with five transcriptional regulator PalRs, each of which is presumed to play a distinctive role in bacterial signaling.

Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in Escherichia coli.

Peptides containing T-cell epitopes from allergens, which are not reactive to allergen-specific IgE, are appropriate candidates as antigens for specific immunotherapy against allergies. To develop a vaccine that can be used in practical application to prevent and treat Japanese cedar pollen allergy, four major T-cell epitopes from the Cry j 1 antigen and six from the Cry j 2 antigen were selected to design cry j 1 epi and cry j 2 epi, DNA constructs encoding artificial polypeptides of the selected epitopes. To apply cholera toxin B subunit (CTB) as an adjuvant, cry j 1 epi and cry j 2 epi were linked and then fused to the CTB gene in tandem to construct a fusion gene, ctb-linker-cry j 1 epi- cry j 2 epi-flag. The fusion gene was introduced into a pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). The expressed recombinant protein was purified by a His-tag affinity column and confirmed by western blot analysis using anti-CTB and anti-FLAG antibodies. The purified recombinant protein also proved to be antigenic against anti-Cry j 1 and anti-Cry j 2 antibodies. Expression of the recombinant protein induced with 1mM IPTG reached a maximum in 3-5h, and recovery of the affinity-purified recombinant protein was approximately 120mg/L of culture medium. The present study indicates that production of sufficient amounts of recombinant protein with antigenic epitopes may be possible by recombinant techniques using E. coli or other bacterial strains for protein expression.

Effects of prodigiosin family compounds from Pseudoalteromonas sp. 1020R on the activities of protein phosphatases and protein kinases.

Pseudoalteromonas sp. strain 1020R produces prodigiosin and its closely related congeners, which differ in the length of their alkyl side chains. These red-pigmented compounds were found to exhibit cytotoxicity against human leukemia cell lines. The compounds also showed dose-dependent inhibitory effects on protein phosphatase 2A and protein tyrosine phosphatase 1B (PTP1B), while remaining relatively inactive against protein kinases, including protein tyrosine kinase, Ca(2+)/calmodulin-dependent protein kinase and protein kinases A and C. Comparative studies of the individual pigmented compounds on PTP1B inhibition showed that as the chain length of the alkyl group at the C-3 position of the compound increased, the inhibitory effect on PTP1B decreased. These results suggest that protein phosphatases but not protein kinases might be involved in the cytotoxicity of the prodigiosin family of compounds against malignant cells.

Draft Genome Sequence of Violacein-Producing Marine Bacterium Pseudoalteromonas sp. 520P1.

Here, we report a draft 5.25-Mb genome sequence of Pseudoalteromonas sp. 520P1, a marine violacein-producing bacterium isolated from the Pacific coast of Japan. Genome annotation by BLAST searches revealed the presence of one acylhomoserine lactone (AHL) synthase (luxI) and five AHL receptor protein (luxR) gene homologs.

Cytotoxic prodigiosin family pigments from Pseudoalteromonas sp. 1020R isolated from the Pacific coast of Japan.

Pseudoalteromonas sp. 1020R, isolated from the Pacific coast of Japan, produces prodigiosin family pigments. Structural analysis indicated that these are prodigiosin (2-methyl-3-pentyl-prodiginine) and three other prodigiosin congeners which differ only in the lengths of the alkyl side chains. These compounds exhibited different extents of cytotoxicity against U937 leukemia cells, and cell death was accompanied by typical features of apoptosis.

Bioactive pigments from marine bacteria: applications and physiological roles.

Research into natural products from the marine environment, including microorganisms, has rapidly increased over the past two decades. Despite the enormous difficulty in isolating and harvesting marine bacteria, microbial metabolites are increasingly attractive to science because of their broad-ranging pharmacological activities, especially those with unique color pigments. This current review paper gives an overview of the pigmented natural compounds isolated from bacteria of marine origin, based on accumulated data in the literature. We review the biological activities of marine compounds, including recent advances in the study of pharmacological effects and other commercial applications, in addition to the biosynthesis and physiological roles of associated pigments. Chemical structures of the bioactive compounds discussed are also presented.

Characterization of a gene cluster and its putative promoter region for violacein biosynthesis in Pseudoalteromonas sp. 520P1.

Violacein, a purple pigment produced by some Gram-negative bacteria, has various physiological properties, such as antitrypanosomal and antitumoral activities. A gene cluster that encodes five enzymes, VioA-VioE, is responsible for synthesizing violacein. The expression of these enzymes is known to be regulated by a quorum sensing mechanism in Chromobacterium violaceum and Pseudoalteromonas sp. 520P1. To clarify the molecular mechanism of regulation of violacein synthesis, we cloned and characterized the gene cluster from Pseudoalteromonas sp. 520P1. A fosmid library of strain 520P1 was constructed and clones containing the gene cluster were isolated. The gene cluster was 7383 bp in length and encoded five enzyme genes, vioA-vioE. A putative promoter sequence was predicted in the upstream region of the cluster. In the promoter region, two contiguous palindromic sequences, a possible quorum sensing regulatory site, were found. However, the isolated Escherichia coli clones harboring the gene cluster and its upstream region were unable to produce violacein probably due to the lack of quorum sensing machinery for expression. To further examine the ability of vioA-vioE genes to synthesize violacein in vivo, the upstream promoter region was removed from the cluster and heterologous expression of the treated cluster was performed in E. coli using a recombinant pET vector with T7 promoter. Purple pigment was expressed, and the pigment was identified to be violacein using ultraviolet and visible light and HPLC analysis. These results will contribute to further studies regarding violacein biosynthesis and its mass production.

Quorum sensing signaling molecules involved in the production of violacein by Pseudoalteromonas.

The production of violacein by Pseudoalteromonas sp. 520P1 has many features of quorum sensing. Signaling molecules were extracted from bacterial culture and subsequently identified as N-(3-oxooctanoyl)-homoserine lactone and N-tetradecanoyl-homoserine lactone. The former but not the latter induced the production of violacein in strain 520P1. We conclude that N-(3-oxooctanoyl)-homoserine lactone is a signaling molecule involved in the production of violacein.

The neuronal circuit of augmenting effects on intrinsic laryngeal muscle activities induced by nasal air-jet stimulation in decerebrate cats.

We previously demonstrated that during nasal air-jet stimulation, both the activities of intrinsic laryngeal adductor and abductor muscles persistently increase, whereas the respiratory cycle prolongs and the activity of diaphragm decreases [Am. J. Rhinol. 9 (1995) 203-208; Neurosci. Res. 31 (1998) 137-146]. The purpose of this study was to clarify the neuronal circuit underlying the augmentation of intrinsic laryngeal muscles evoked by nasal air-jet stimulation. The immunohistologic analysis of Fos-expression was reported to determine the distribution of activated neurons in cat brainstem evoked by sneeze-inducing air puff stimulation of the nasal mucosa [Brain Res. 687 (1995) 143-154]. In sneezing cats, immunoreactivity was evoked in projection areas of the ethmoidal afferents, e.g. the subnuclei caudalis, interpolaris and in interstitial islands of the trigeminal sensory complex. Immunoreactivity was also enhanced in the solitary complex, the nucleus retroambiguus, the pontine parabrachial area and the lateral aspect of the parvocellular reticular formation [Brain Res. 687 (1995) 143-154]. In the present study, we focussed on the parvocellular reticular nucleus (PRN) as a relay of the neural circuit contributed to the augmentation of intrinsic laryngeal muscles evoked by nasal air-jet stimulation. We recorded the neuronal behavior of PRN during the nasal air-jet stimulation in precollicular-postmammillary decerebrate cats. As the results, 24% (17/71) of recorded neurons which were activated orthodromically by the electrical stimulation to anterior ethmoidal nerve, increased their firing rates in response to the nasal air-jet stimulation. Furthermore, spike-triggered averaging method revealed that four of these 17 PRN neurons activated intrinsic laryngeal muscles, suggesting that such neurons have excitatory projections to the intrinsic laryngeal muscle motoneurons in the nucleus ambiguus. These results suggest that the some of PRN neuron play a role in augmentation of the intrinsic laryngeal muscles activities during nasal air-jet stimulation.