Cloning and expression of protective antigens of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Ag85B and ESAT-6 in <Emphasis Type="Italic">Francisella tularensis</Emphasis> 15/10

The possibility of expression of genes encoding mycobacterial antigens in Francisella tularensis 15/10 vaccine strain cells has been shown for the first time. To obtain stable and effective expression of mycobacterial antigens in the F. tularensis cells, the plasmid vector pPMC1 and hybrid genes consisting of the leader part FL of the F. tularensis membrane protein FopA and structural moieties of the mature protein Ag85B or the fused protein Ag85B-ESAT-6 were constructed. Recombinant strains F. tularensis RVp17 and RVp18 expressing protective mycobacterial antigens in the fused proteins FL-Ag85B and FL-Ag85B-ESAT-6, respectively, were obtained. Expression of the protective mycobacterial antigens in F. tularensis was analyzed using specific antisera to the recombinant proteins Ag85-(His)6 and ESAT-6-(His)6 isolated from Escherichia coli producer strains created on the basis of the pET23b(+) and pET24b(+) vectors. The expression of heterologous protective antigens in F. tularensis 15/10 is promising for creation of live recombinant anti-tuberculosis vaccines on the basis of the tularemia vaccine strain.     

Genetic analysis of innate immunity: TIR adapter proteins in innate and adaptive immune responses

The innate immune system senses pathogens largely through signals initiated by a collection of phylogenetically related proteins known as "Toll-like receptors" (TLRs), of which 10 representatives are encoded in the human genome. Our understanding of the sensing role played by the TLRs began with the positional cloning of a spontaneous mutation (Lps(d)) in the gene encoding the mammalian lipopolysaccharide (LPS) receptor. Other key innate immunity proteins have been disclosed by germline mutagenesis, and are discussed in the present review.     

Identification of a novel Francisella tularensis factor required for intramacrophage survival and subversion of innate immune response

Francisella tularensis, the causative agent of tularemia, is one of the deadliest agents of biological warfare and bioterrorism. Extremely high virulence of this bacterium is associated with its ability to dampen or subvert host innate immune response. The objectives of this study were to identify factors and understand the mechanisms of host innate immune evasion by F. tularensis. We identified and explored the pathogenic role of a mutant interrupted at gene locus FTL_0325, which encodes an OmpA-like protein. Our results establish a pathogenic role of FTL_0325 and its ortholog FTT0831c in the virulent F. tularensis SchuS4 strain in intramacrophage survival and suppression of proinflammatory cytokine responses. This study provides mechanistic evidence that the suppressive effects on innate immune responses are due specifically to these proteins and that FTL_0325 and FTT0831c mediate immune subversion by interfering with NF-κB signaling. Furthermore, FTT0831c inhibits NF-κB activity primarily by preventing the nuclear translocation of p65 subunit. Collectively, this study reports a novel F. tularensis factor that is required for innate immune subversion caused by this deadly bacterium.     

Injection of xenogeneic cells into teleost fish elicits systemic and local cellular innate immune responses

The early innate immune response of the teleost gilthead seabream (Sparus aurata L.) against xenogeneic cells was studied. Fish received a single intraperitoneal injection of xenogeneic cells (tumour cell line), following which leucocyte mobilization, degranulation, peroxidase content, respiratory burst and phagocytic and cytotoxic activities were determined in both peritoneal exudate leucocytes (PELs) and head-kidney leucocytes (HKLs). The total number of PELs increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis of PEL and HKL suspensions revealed variations in the proportion of cell types. The percentage of HK acidophilic granulocytes significantly increased after 72 h, whereas PE acidophils increased after 4 h. Moreover, numbers of PE lymphocytes and monocyte-macrophages significantly increased during the experiment. The peroxidase content of the leucocytes was unaffected, although PEL degranulation was largely enhanced. This liberation of peroxidases correlated well with the enhancement of the oxidative respiratory burst activity in PELs, reflecting leucocyte activation. However, phagocytosis only increased in PELs 4 h after intraperitoneal injection, whereas the cytotoxic activity of HKLs increased 1 and 2 days post-injection but, in general, decreased in the PELs. Our data thus demonstrate that the appearance of xenogeneic cells involves leucocyte mobilization and innate immune-response activation at the site of invasion and in the head-kidney. Involvement of the various leucocyte types and potential modes of activation are discussed.This work was partially funded by the European Commission (QLRT-2001-00722). A. Cuesta and I. Salinas are fellows of Fundación CajaMurcia and Fundación Séneca, respectively.     

Allelic variation at the linked AP1 and PhyC loci in hexaploid wheat is associated but not perfectly correlated with vernalization response

Vernalization requirement is an important trait in temperate crop plants such as wheat and must be considered when selecting varieties for cultivation under different climatic conditions. To determine the growth habit of wheat varieties, plants need to be grown under different vernalization regimes, a lengthy but necessary process for breeders involved in crossing winter with spring germplasm. If haplotypes can be associated with growth habit, then molecular marker assays that are reliable, cheap, and quick can be developed to assist in the selection of plants with the desired phenotype. We have analyzed 81 accessions that have different vernalization requirements and putative different origins of spring habit for sequence variation at the Apetala1 (AP1) locus, which underlies Vrn-1, and at the linked Phytochrome C (PhyC) locus. Good correspondence was found between the AP1 genotype and the PhyC haplotype for 77 of the 81 accessions. Two varieties displayed a recombination event between the AP1 and PhyC loci, and one variety carried a recombinant PhyC gene. In addition, one variety carried an apparent AP1 winter allele, but displayed the Vrn-A1 spring habit. The PhyC haplotype for this variety also indicated the presence of a Vrn-A1 spring allele. Our data suggest that both the AP1 promoter region and PhyC SNPs can be used as diagnostic markers for vernalization response at the vrn-A1 locus, but that neither are perfect tags.     

The N-glycolyl form of mouse sialyl Lewis X is recognized by selectins but not by HECA-452 and FH6 antibodies that were raised against human cells

E-, P- and L-selectins critically function in lymphocyte recirculation and recruiting leukocytes to inflammatory sites. MECA-79 antibody inhibits L-selectin-mediated lymphocyte adhesion in several species and does not require sialic acid in its epitope. Many other antibodies, however, recognize human selectin ligands expressing N-acetylneuraminic acid but not mouse selectin ligands expressing N-glycolylneuraminic acid, suggesting that difference in sialic acid in sialyl Lewis X leads to differential reactivity. We found that HECA-452 and FH6 monoclonal antibodies bind Chinese hamster ovary (CHO) cells expressing N-acetylneuraminyl Lewis X oligosaccharide but not its N-glycolyl form. Moreover, synthetic N-acetylneuraminyl Lewis X oligosaccharide but not its N-glycolyl oligosaccharide inhibited HECA-452 and FH6 binding. By contrast, E-, P- and L-selectin bound to CHO cells regardless of whether they express N-acetyl or N-glycolyl form of sialyl Lewis X, showing that selectins have a broader recognition capacity than HECA-452 and FH-6 anti-sialyl Lewis x antibodies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection

Eimeria spp. are the causative agents of coccidiosis, a major disease affecting the poultry industry. A recombinant non-antibiotic Escherichia coli that expresses the Eimeria tenella SO7 gene was constructed and its protective efficacy against homologous infection in chickens was determined. The three-day-old chickens were orally immunized with the recombinant non-antibiotic SO7 gene expressing E. coli and boosted two weeks later. Four weeks after the second immunization, the chickens were challenged with 5 × 10⁴ homologous sporulated oocysts. The protective effects of the recombinant non-antibiotic E. coli were determined by measuring body weight change, mortality, histopathology, lesion scores, oocyst counts, the specific antibody response and the frequency of CD4⁺ and CD8⁺ lymphocytes in peripheral blood. The results showed that immunization with SO7 expressing E. coli resulted in significantly improved body weight gain, reduced lesion scores and oocyst shedding in immunized chickens compared to controls. Furthermore, administration of recombinant SO7 expressing E. coli leads to a significant increase in serum antibody, CD4⁺ and CD8⁺ T cells in peripheral blood of chickens. These results, therefore, suggest that the recombinant non-antibiotic E. coli that expresses the SO7 gene is able to effectively stimulate host protective immunity as evidenced by the induction of development of both humoral and cell-mediated immune responses against homologous challenge in chickens.     

Inv1: an Edwardsiella tarda invasin and a protective immunogen that is required for host infection

Invasin is an outer membrane protein that is known to mediate entry of enteric bacteria into mammalian cells. In this study, we analyzed the function and immunoprotective potential of the invasin Inv1 from Edwardsiella tarda, a serious fish pathogen that can also infect humans. In silico analysis indicated that Inv1 possesses a conserved N-terminal DUF3442 domain and a C-terminal group 1 bacterial Ig-like domain. Subcellular localization analysis showed that Inv1 is exposed on cell surface and could be recognized by specific antibodies. Mutation of inv1 had no effect on bacterial growth but attenuates overall bacterial virulence and impaired the ability of E. tarda to attach and invade into host cells. Consistent with these observations, antibody blocking of Inv1 inhibited E. tarda infection of host cells. To examine the immunoprotective potential of Inv1, recombinant Inv1 (rInv1) corresponding to the DUF3442 domain was purified and used to vaccinate Japanese flounder (Paralichthys olivaceus). The results showed that rInv1 induced strong protection against lethal-dose challenge of E. tarda. ELISA analysis showed that rInv1-vaccinated fish produced specific serum antibodies that could enhance the serum bactericidal activity against E. tarda. Taken together, these results indicate that Inv1 is a surface-localized virulence factor that is involved in host infection and can induce effective immunoprotection when used as a subunit vaccine.     

High susceptibility to respiratory Acinetobacter baumannii infection in A/J mice is associated with a delay in early pulmonary recruitment of neutrophils

Acinetobacter baumannii is an important cause of both community-associated and nosocomial pneumonia, which have become increasingly difficult to treat because of the rapid development of resistance to multiple antibiotics. Despite its clinical importance, the pathogenesis of and host defense against respiratory A. baumannii infection remains largely unknown. To examine host factors that could contribute to the defense, we compared the susceptibilities of A/J and C57BL/6 mice to intranasal (i.n.) inoculation with A. baumannii. We found that A/J mice were significantly more susceptible to infection with higher mortality (P < 0.05) and tissue bacterial burdens (P < 0.01) as well as greater histopathology in the lung and spleen than C57BL/6 mice. More importantly, the high susceptibility of A/J mice was associated with a reduced local proinflammatory cytokine/chemokine (particularly IL-1β, MIP-2 and TNF-α) responses and a significant delay and reduction in the early influx of neutrophils in the lung (P < 0.05). Intranasal administration of neutrophil-inducing chemokine MIP-2 to A/J mice enhanced pulmonary neutrophil influx and partially restored host resistance to A. baumannii to a level comparable to the more resistant C57BL/6 mice. Our results imply that the early recruitment of neutrophils into the lung is critical for initiating an efficient host defense against respiratory A. baumannii infection.     

Molecular analysis of the Chlamydomonas nuclear gene encoding PsbW and demonstration that PsbW is a subunit of photosystem II,but not photosystem I

PsbW is a nuclear-encoded protein located in the thylakoid membrane of the chloroplast. Studies in higher plants have provided substantial evidence that PsbW is a core component of photosystem II. However, recent data have been presented to suggest that PsbW is also a subunit of photosystem I. Such a sharing of subunits between the two photosystems would represent a novel phenomenon. To investigate this, we have cloned and characterized the psbW gene from the green alga Chlamydomonas reinhardtii. The gene is split by five introns and encodes a polypeptide of 115 residues comprising the 6.1 kDa mature PsbW protein preceded by a 59 amino acid bipartite transit sequence. Using antibodies raised to PsbW we have examined: (1) C. reinhardtii mutants lacking either photosystem and (2) purified photosystem preparations. We find that PsbW is a subunit of photosystem II, but not photosystem I.     

Plasmodium: Mammalian codon optimization of malaria plasmid DNA vaccines enhances antibody responses but not T cell responses nor protective immunity

We have evaluated the effect of mammalian codon optimization on the immunogenicity and protective efficacy of plasmid DNA vaccines encoding pre-erythrocytic stage Plasmodium falciparum and Plasmodium yoelii antigens in mice. Codon optimization significantly enhanced in vitro expression and in vivo antibody responses for P. falciparum circumsporozoite protein (PfCSP) and P. yoelii hepatocyte erythrocyte protein 17 kDa (PyHEP17) but not for P. yoelii circumsporozoite protein (PyCSP). Unexpectedly, more robust CD4⁺ and CD8⁺ T cell responses as measured by IFN-γ ELIspot, lymphoproliferation, and cytotoxic T lymphocyte assays were noted with native as compared with codon optimization constructs. Codon optimization also failed to enhance CD8⁺ T cell dependent protection against P. yoelii sporozoite challenge as measured by liver-stage parasite burden. These data demonstrate that the effect of mammalian codon optimization is antigen-dependent and may not be beneficial for vaccines designed to induce T cell dependent protective immunity in this malaria model.     

Examination of the immune responses of males and workers of the leaf-cutting ant Acromyrmex echinatior and the effect of infection

Summary. Parasites represent significant challenges to social insects. The high density, interaction rate and relatedness of individuals within colonies are all predicted to make social insect colonies particularly vulnerable to parasites. To cope with this pressure, social insects have evolved a number of defence mechanisms. These include the immune response, which, aside from in bumblebees, has been relatively little studied in social insects. Here we compare the immune responses of males and workers of the leaf-cutting ant Acromyrmex echinatior and examine the effect upon immunocompetence of prior exposure to a virulent parasite. Males have a far lower immune response than workers, suggesting either haploid susceptibility or reduced investment in immunity by the short-lived males. There was also significantly less variation in the immune response of males than of workers, which may be due to leaf-cutting ant workers being more variable in age or more genetically diverse within colonies. When exposed to the entomopathogenic fungus Metarhizium, workers expressed a substantially reduced immune response 96 h after infection, suggesting that the immune system was either depleted by having to respond to the Metarhizium infection or was depressed by the parasite. The results suggest that the immune response is a costly and limited process, but further experiments are needed to distinguish between the alternative explanations for the effects observed.Received 3 August 2004; revised 3 February 2005; accepted 2 March 2005.     

Induced defence responses and protective effects on tomato against Xanthomonas vesicatoria by an aqueous extract from Solanum lycocarpum infected with Crinipellis perniciosa

This study investigated the induced defence responses and protective effects on susceptible tomato (Lycopersicon esculentum Mill.) against Xanthomonas vesicatoria (Doidge) by a heat-treated aqueous extract (VLA) from dry necrotic tissue of ‘Lobeira’ (Solanum lycocarpum St. Hil.) branches infected with the fungus Crinipellis perniciosa (Stahel) compared with acibenzolar-S-methyl (ASM), a commercial inducer of resistance. Plantlets were sprayed with VLA and ASM and challenged 4 days later with a virulent strain of X. vesicatoria, under greenhouse conditions. The disease severity, fresh weight of shoots, the activities of phenol peroxidase (POX), polyphenol oxidase (PPO), chitinase (CHI), phenylalanine ammonia-lyase (PAL), lignin deposition, and soluble phenolic contents were evaluated in the leaf tissues. Reduction of the bacterial spot severity was observed in plantlets treated with VLA which conferred 63% of the ASM protection. This protective effect and lesion reduction promoted by VLA were probably associated particularly with POX and PAL activities, lignin deposition on leaf tissues and, to a less extent, CHI activity.     

A rice serine carboxypeptidase-like gene OsBISCPL1 is involved in regulation of defense responses against biotic and oxidative stress

Serine carboxypeptidase-like proteins (SCPLs) comprise a large family of protein hydrolyzing enzymes that play roles in multiple cellular processes. During the course of study aimed at elucidating the molecular basis of induced immunity in rice, a gene, OsBISCPL1, encoding a putative SCPL, was isolated and identified. OsBISCPL1 contains a conserved peptidase S10 domain, serine active site and a signal peptide at N-terminus. OsBISCPL1 is expressed ubiquitously in rice, including roots, stems, leaves and spikes. Expression of OsBISCPL1 in leaves was significantly up-regulated after treatments with benzothiadiazole, salicylic acid, jasmonic acid and 1-amino cyclopropane-1-carboxylic acid, and also up-regulated in incompatible interactions between rice and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants with constitutive expression of OsBISCPL1 were generated and disease resistance assays indicated that the OsBISCPL1-overexpressing plants showed an enhanced disease resistance against Pseudomonas syringae pv. tomato and Alternaria brassicicola. Expression levels of defense-related genes, e.g. PR1, PR2, PR5 and PDF1.2, were constitutively up-regulated in transgenic plants as compared with those in wild-type plants. Furthermore, the OsBISCPL1-overexpressing plants also showed an increased tolerance to oxidative stress and up-regulated expression of oxidative stress-related genes. The results suggest that the OsBISCPL1 may be involved in regulation of defense responses against pathogen infection and oxidative stress.     

The type-1 and type-2 ribosome-inactivating proteins from<Emphasis Type="Italic"> Iris</Emphasis> confer transgenic tobacco plants local but not systemic protection against viruses

The antiviral activity of the type-2 ribosome-inactivating protein (RIP) IRAb from Iris was analyzed by expressing IRAb in tobacco (Nicotiana tabacum L. cv. Samsun NN) plants and challenging the transgenic plants with tobacco mosaic virus (TMV). Although constitutive expression of IRAb resulted in an aberrant phenotype, the plants were fertile. Transgenic tobacco lines expressing IRAb showed a dose-dependent enhanced resistance against TMV infection but the level of protection was markedly lower than in plants expressing IRIP, the type-1 RIP from Iris that closely resembles the A-chain of IRAb. To verify whether IRIP or IRAb can also confer systemic protection against viruses, transgenic RIP-expressing scions were grafted onto control rootstocks and leaves of the rootstocks challenged with tobacco etch virus (TEV). In spite of the strong local antiviral effect of IRIP and IRAb the RIPs could not provide systemic protection against TEV. Hence our results demonstrate that expression of the type-1 and type-2 RIPs from Iris confers tobacco plants local protection against two unrelated viruses. The antiviral activity of both RIPs was not accompanied by an induction of pathogenesis-related proteins. It is suggested that the observed antiviral activity of both Iris RIPs relies on their RNA N-glycohydrolase activity towards TMV RNA and plant rRNA.Abbreviations GUS -Glucuronidase - IRAb Iris agglutinin b - IRIP Iris type-1 RIP - PAG Polynucleotide:adenosine glycosylase - PAP Phytolacca americana antiviral protein - PR Pathogenesis-related - RIP Ribosome-inactivating protein - TCS Trichosanthin - TEV Tobacco etch virus - TMV Tobacco mosaic virus     

Over-expression of OsUGE-1 altered raffinose level and tolerance to abiotic stress but not morphology in Arabidopsis

OsUGE-1 is known to be induced by various abiotic stresses, but its exact function in plants is unclear. In the present study, OsUGE-1 was over-expressed in Arabidopsis, transgenic plants conferred tolerance to salt, drought and freezing stress without altering plant morphology. In addition, transgenic plants showed a higher level of the soluble sugar raffinose than did wild-type plants. Our results suggest that elevated level of raffinose with over-expressed OsUGE-1 resulted in enhanced tolerance to abiotic stress. Thus, the gene may be applied to improve tolerance to abiotic stress in crops.     

Fusion of circular and longitudinal muscles in Drosophila is independent of the endoderm but further visceral muscle differentiation requires a close contact between mesoderm and endoderm

In this study we describe the morphological and genetic analysis of the Drosophila mutant gürtelchen (gurt). gurt was identified by screening an EMS collection for novel mutations affecting visceral mesoderm development and was named after the distinct belt shaped visceral phenotype. Interestingly, determination of visceral cell identities and subsequent visceral myoblast fusion is not affected in mutant embryos indicating a later defect in visceral development. gurt is in fact a new huckebein (hkb) allele and as such exhibits nearly complete loss of endodermal derived structures. Targeted ablation of the endodermal primordia produces a phenotype that resembles the visceral defects observed in huckebein^gürtelchen (hkb^gurt) mutant embryos.It was shown previously that visceral mesoderm development requires complex interactions between visceral myoblasts and adjacent tissues. Signals from the neighbouring somatic myoblasts play an important role in cell type determination and are a prerequisite for visceral muscle fusion. Furthermore, the visceral mesoderm is known to influence endodermal migration and midgut epithelium formation. Our analyses of the visceral phenotype of hkb^gurt mutant embryos reveal that the adjacent endoderm plays a critical role in the later stages of visceral muscle development, and is required for visceral muscle elongation and outgrowth after proper myoblast fusion.     

Interaction of cruciferous phytoanticipins with plant fungal pathogens: indole glucosinolates are not metabolized but the corresponding desulfo-derivatives and nitriles are

Glucosinolates represent a large group of plant natural products long known for diverse and fascinating physiological functions and activities. Despite the relevance and huge interest on the roles of indole glucosinolates in plant defense, little is known about their direct interaction with microbial plant pathogens. Toward this end, the metabolism of indolyl glucosinolates, their corresponding desulfo-derivatives, and derived metabolites, by three fungal species pathogenic on crucifers was investigated. While glucobrassicin, 1-methoxyglucobrassicin, 4-methoxyglucobrassicin were not metabolized by the pathogenic fungi Alternaria brassicicola, Rhizoctonia solani and Sclerotinia sclerotiorum, the corresponding desulfo-derivatives were metabolized to indolyl-3-acetonitrile, caulilexin C (1-methoxyindolyl-3-acetonitrile) and arvelexin (4-methoxyindolyl-3-acetonitrile) by R. solani and S. sclerotiorum, but not by A. brassicicola. That is, desulfo-glucosinolates were metabolized by two non-host-selective pathogens, but not by a host-selective. Indolyl-3-acetonitrile, caulilexin C and arvelexin were metabolized to the corresponding indole-3-carboxylic acids. Indolyl-3-acetonitriles displayed higher inhibitory activity than indole desulfo-glucosinolates. Indolyl-3-methanol displayed antifungal activity and was metabolized by A. brassicicola and R. solani to the less antifungal compounds indole-3-carboxaldehyde and indole-3-carboxylic acid. Diindolyl-3-methane was strongly antifungal and stable in fungal cultures, but ascorbigen was not stable in solution and displayed low antifungal activity; neither compound appeared to be metabolized by any of the three fungal species. The cell-free extracts of mycelia of A. brassicicola displayed low myrosinase activity using glucobrassicin as substrate, but myrosinase activity was not detectable in mycelia of either R. solani or S. sclerotiorum.