Immune response of rats vaccinated orally with various plant-expressed recombinant cysteine proteinase constructs when challenged with Fasciola hepatica metacercariae.

BACKGROUND: Cysteine proteinases of Fasciola hepatica are important candidates for vaccine antigens because of their role in fluke biology and host-parasite relationships. In our previous experiments, we found that a recombinant cysteine proteinase cloned from adult F. hepatica (CPFhW) can protect rats against liver fluke infections when it is administered intramuscularly or intranasally in the form of cDNA. We also observed considerable protection upon challenge following mucosal vaccination with inclusion bodies containing recombinant CPFhW produced in Escherichia coli. In this study, we explore oral vaccination, which may be the desired method of delivery and is potentially capable of preventing infections at the site of helminth entry. To provide antigen encapsulation and to protect the vaccine antigen from degradation in the intestinal tract, transgenic plant-based systems are used. METHODOLOGY: In the present study, we aimed to evaluate the protective ability of mucosal vaccinations of 12-week-old rats with CPFhW produced in a transgenic-plant-based system. To avoid inducing tolerance and to maximise the immune response induced by oral immunisation, we used the hepatitis B virus (HBV) core protein (HBcAg) as a carrier. Animals were immunised with two doses of the antigen and challenged with 25 or 30 metacercariae of F. hepatica. CONCLUSIONS: We obtained substantial protection after oral administration of the plant-produced hybrids of CPFhW and HBcAg. The highest level of protection (65.4%) was observed in animals immunised with transgenic plants expressing the mature CPFhW enzyme flanked by Gly-rich linkers and inserted into c/e1 epitope of truncated HBcAg. The immunised rats showed clear IgG1 and IgM responses to CPFhW for 4 consecutive weeks after the challenge.

Ferritins and nodulation in Lupinus luteus: iron management in indeterminate type nodules.

An ability to form symbiotic associations with rhizobia and to utilize atmospheric nitrogen makes legumes ecologically successful. High iron content in legume grains, partially relocated from root nodules, is another-nutritional-advantage of this group of plants. The ferritin complex is the major cell iron storage and detoxification unit and has been recognized as a marker of many stress-induced responses. The possible participation of ferritin in nodule formation and functioning was investigated here. Correlation of increased accumulation of both ferritin polypeptide and mRNA with actual in situ localization of ferritin allowed ferritin synthesis in the developing, indeterminate-type root nodules to be related to differentiating bacteroid tissue. This kind of tissue, in contrast to the determinate-type nodules, is present in lupin nodules at almost all stages of their development. Interestingly, it was found that, in this type of nodule, senescence starting in the decaying zones induces ferritin accumulation in younger, still active, tissues. Based on the presented data, and in correlation with previous results, some aspects of the regulation of expression of lupin ferritin genes are also discussed.

Immunoprotective properties of transgenic plants expressing E2 glycoprotein from CSFV and cysteine protease from Fasciola hepatica.

Immune responses were elicited in laboratory animals after oral vaccination by transgenic plants (lettuce and alfalfa) expressing the E2 glycoprotein of Classical Swine Fever Virus (CSFV) or cysteine protease from Fasciola hepatica. ELISA analyses demonstrated that the oral route is effective in inducing a specific antibody response against these antigens in mice.

Bayesian phylogenetic analysis reveals two-domain topology of S-adenosylhomocysteine hydrolase protein sequences.

S-Adenosylhomocysteine hydrolase (SahH) is involved in the degradation of the compound which inhibits methylation reactions. Using a Bayesian approach and other methods, we reconstructed a phylogenetic tree of amino acid sequences of this protein originating from all three major domains of living organisms. The SahH sequences formed two major branches: one composed mainly of Archaea and the other of eukaryotes and majority of bacteria, clearly contradicting the three-domain topology shown by small subunit rRNA gene. This topology suggests the occurrence of lateral transfer of this gene between the domains. Poor resolution of eukaryotes and bacteria excluded an ultimate conclusion in which out of the two domains this gene appeared first, however, the congruence of the secondary branches with SS rRNA and/or concatenated ribosomal protein datasets phylogenies suggested an "early" acquisition by some bacterial and eukaryotic phyla. Similarly, the branching pattern of Archaea reflected the phylogenies shown by SS rRNA and ribosomal proteins. SahH is widespread in Eucarya, albeit, due to reductive evolution, it is missing in the intracellular parasite Encephalitozoon cuniculi. On the other hand, the lack of affinity to the sequences from the alpha-Proteobacteria and cyanobacteria excludes a possibility of its acquisition in the course of mitochondrial or chloroplast endosymbioses. Unlike Archaea, most bacteria carry MTA/SAH nucleosidase, an enzyme involved also in metabolism of methylthioadenosine. However, the double function of MTA/SAH nucleosidase may be a barrier to ensure the efficient degradation of S-adenosylhomocysteine, specially when the intensity of methylation processes is high. This would explain the presence of S-adenosylhomocysteine hydrolase in the bacteria that have more complex metabolism. On the other hand, majority of obligate pathogenic bacteria due to simpler metabolism rely entirely on MTA/SAH nucleosidase. This could explain the observed phenetic pattern in which bacteria with larger (>6 Mb-million base pairs) genomes carry SAH hydrolase, whereas bacteria that have undergone reductive evolution usually carry MTA/SAH nucleosidase. This suggests that the presence or acquisition of S-adenosylhomocysteine hydrolase in bacteria may predispose towards higher metabolic, and in consequence, higher genomic complexity. The good examples are the phototrophic bacteria all of which carry this gene, however, the SahH phylogeny shows lack of congruence with SSU rRNA and photosyntethic genes, implying that the acquisition was independent and presumably preceded the acquisition of photosyntethic genes. The majority of cyanobacteria acquired this gene from Archaea, however, in some species the sahH gene was replaced by a copy from the beta- or gamma-Proteobacteria.

Low sequence similarity and gene content of symbiotic clusters of Bradyrhizobium sp. WM9 (Lupinus) indicate early divergence of "lupin" lineage in the genus Bradyrhizobium.

Two sequenced nodulation regions of lupin Bradyrhizobium sp. WM9 carried the majority of genes involved in the Nod factor production. The nod region I harbored: nolA, nodD, nodA, nodB, nodC, nodS, nodI, nodJ, nolO, nodZ, fixR, nifA, fixA, nodM, nolK and noeL. This gene arrangement resembled that found in the nodulation region of Bradyrhizobium japonicum USDA110, however strain WM9 harbored only one nodD gene copy, while the nodM, nolK and noeL genes had no counterparts in the 410 kb symbiotic region of strain USDA110. Region II harbored nolL and nodW, but lacked an nodV gene. Both regions carried ORFs that lacked similarity to the published USDA110 sequences, though they had homologues in symbiotic regions of Rhizobium etli, Sinorhizobium sp. NGR234 and Mesorhizobium loti. These differences in gene content, as well as a low average sequence identity (70%) of symbiotic genes with respect to B. japonicum USDA110 were in contrast with the phylogenetic relationship of USDA110 and WM9 revealed by the analysis of 16S rDNA and dnaK sequences. This most likely reflected an early divergence of symbiotic loci, and possible co-speciation with distinct legumes. During this process the loss of a noel gene and the acquisition of a nolL gene could be regarded as an adaptation towards these legumes that responded to Nod factors carrying 4-O-acetylfucose rather than 2-O-methylfucose. This explained various responses of lupins and serradella plants to infection by mutants in nodZ and nolL genes, knowing that serradella is a stringent legume while lupins are more promiscuous legumes.

Yellow lupine gene encoding stearoyl-ACP desaturase--organization, expression and potential application.

A gene for the delta9 desaturase specific to stearoyl-ACP (acyl carrier protein) was identified from yellow lupine (Lupinus luteus) cDNA and genomic libraries through the differential display method. The desaturase transcript appears in plants infected with Bradyrhizobium sp. (Lupinus) as revealed by Northern hybridization, RT-PCR and expression of beta-glucuronidase under the desaturase promoter. A small amount of desaturase transcript was also detected in uninfected plants, which suggests that the gene does not belong to the strict nodule-specific sequences. The desaturase provides unsaturated fatty acids for additional cell membrane synthesis. During nodule and symbiosome development a peribacteroid membrane is formed and the requirement for membrane surface increases, thus the level of desaturase expression is also higher. Transgenic plants of Nicotiana tabacum with overexpression of the full-length lupine stearoyl-ACP desaturase sequence were obtained. They revealed higher content of unsaturated fatty acids (especially oleic acid) in comparison with control plants.