Induction of secretion and surface capping of microneme proteins in Eimeria tenella

Micronemes are secretory organelles of the invasive stages of apicomplexan parasites and contain proteins that are important for parasite motility and host cell invasion. We have examined the induction of microneme secretion in the coccidian Eimeria tenella. When sporozoites were added to MDBK cells in culture, microneme proteins were secreted, capped backwards over the parasite surface and deposited onto underlying host cells from the posterior end of gliding parasites. Induction of secretion was also achieved by the addition of foetal calf serum, or purified albumin, to extracellular sporozoites. Microneme secretion per se was not dependent on parasites being able to move or to invade host cells. However, in the presence of cytochalasin D, which disrupts actin polymerisation and prevents parasite movement, microneme proteins were secreted from the apical tip but were not capped backwards over the sporozoite surface. These observations support the hypothesis that microneme proteins function as ligands which, when secreted out onto the parasite surface, form a link, either directly or indirectly, between the sub-pellicular actin–myosin cytoskeletal motor of the parasite and the surface of target host cells.     

Identification and characterization of cDNA clones encoding antigens of Eimeria tenella

An Eimeria tenella cDNA library was constructed in the expression vector λgt11 from poly (A⁺) RNA extracted from sporulating oocysts. The library was screened with rabbit antiserum raised against antigens extracted from fully sporulated oocysts. All of the antigen-expressing plaque-purified clones were initially characterized by cross screening with antisera raised against different stages of the E. tenella life cycle, as well as with antiserum raised against sporozoites of a related species, namely E. acervulina. A selected number of clones were further characterized by antibody selection coupled with immunoblotting and DNA cross hybridization. Three different E. tenella antigens were identified. All three appear to be constitutively expressed at the protein level during sporogony.     

Extracellular calcium deficiency and ryanodine inhibit Eimeria tenella sporozoite invasion in vitro

An in vitro assay system with Eimeria tenella sporozoites was used to study the effects of extracellular calcium and active agents affecting the invasion of parasites into host cells. At concentrations of 900M Ca²⁺ and less the invasion rates were distinctly decreased. Ryanodine, a herbal alkaloid known for binding to internal Ca²⁺ channels (ryanodine receptors), showed an inhibitory effect on E. tenella sporozoite invasion. Preincubation tests and staining with a fluorescent derivative of ryanodine assured that the compound bound specifically to the sporozoites and affected them rather than the host cells.All experiments described in this publication comply with the current laws of Germany     

Synergistic interaction between the Potyvirus, Turnip mosaic virus and the Crinivirus, Lettuce infectious yellows virus in plants and protoplasts

Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, is specifically transmitted by the sweet potato whitefly (Bemisia tabaci) in a semipersistent manner. LIYV infections result in a low virus titer in plants and protoplasts, impeding reverse genetic efforts to analyze LIYV gene/protein functions. We found that synergistic interactions occurred in mixed infections of LIYV and Turnip mosaic virus (TuMV) in Nicotiana benthamiana plants, and these resulted in enhanced accumulation of LIYV. Furthermore, we examined the ability of transgenic plants and protoplasts expressing only the TuMV P1/HC-Pro sequence to enhance the accumulation of LIYV. LIYV RNA and protein titers increased by as much as 8-fold in these plants and protoplasts relative to control plants. LIYV infections remained phloem-limited in P1/HC-Pro transgenic plants, suggesting that enhanced accumulation of LIYV in these plants was due primarily to increased replication efficiency, not to greater spread.     

Molecular characterization of a coccidian parasite cGMP dependent protein kinase

The cGMP-dependent protein kinase (PKG) of Eimeria tenella and Toxoplasma gondii is the target of a novel coccidiostat that is effective against coccidiosis and toxoplasmosis in animal models. Preparations of native PKG enzyme from Toxoplasma and Eimeria contain a membrane-associated polypeptide (isoform-I) of about 110 kDa and a slightly smaller soluble polypeptide (isoform-II). Expression of T. gondii and E. tenella PKG cDNA clones in Toxoplasma yield similarly sized recombinant polypeptides, which co-migrate on SDS-polyacrylamide gels with the corresponding native isoforms. Results of targeted mutagenesis of potential translational initiation sites suggest that parasite isoform-II is a product of alternative translational initiation from an internal initiator methionine codon. Exclusive expression of isoform-II or isoform-I can be achieved by preventing initiation at the respective primary or secondary sites. Immunofluorescence analysis indicates that recombinant isoform-I localizes primarily to the parasite plasma membrane, while isoform-II remains cytosolic. Mutagenesis and metabolic labeling studies reveal that the observed membrane-association of full-length recombinant PKG is mediated by N-terminal myristoylation and palmitoylation at amino acids G2 and C4. We also confirm the functional significance of a putative third PKG allosteric site, common to apicomplexan PKGs but absent from vertebrate or insect PKGs. In assays with transiently transfected parasites, constructs harboring a mutation at this site express markedly lower levels of cGMP-dependent PKG activity, while a triple mutant bearing mutations in all three sites reduces kinase activity to background levels.     

TLR2 and TLR4 polymorphisms in familial Mediterranean fever

It has been suggested that MEVF mutations offer advantage against infections, including tuberculosis. Bearing in mind the central role of TLR-2 and TLR-4 in the recognition of pathogens, we conducted this study to examine whether the TLR2-R753Q, TLR4-D299G, TLR4-T399I common polymorphisms are associated with susceptibility to familial Mediterranean fever (FMF) or affect the course of the disease. A cohort of 169 FMF patients and 245 healthy bone marrow donors were enrolled in the study. FMF patients appeared with a significantly lower frequency of the TLR4-D299G mutated allele (3.2% vs 6.9%, p = 0.032). No association was observed with the other analyzed polymorphisms. Moreover, we found no association between polymorphisms and the frequency of attacks or the development of amyloidosis. Our results may reinforce the hypothesis that FMF patients display a better defense against pathogens, providing an additional mechanism and suggesting a positive selection advantage in the area of the Mediterranean basin.     

Non-opsonic attachment of Bordetella bronchiseptica mediated by CD11/CD18 and cell surface carbohydrates

Porcine atrophic rhinitis associated with Bordetella bronchiseptica is characterized by a severe inflammatory response in the mucosa of the nasal turbinates. Initial infiltrates of polymorphonuclear leukocytes (PMN) are followed by accumulations of mononuclear cells. In this report, we have investigated the interaction between porcine PMN and B. bronchiseptica. PMN incubated in PBS with a fluorescently labeled hemagglutinating porcine isolate, but not a non-hemagglutinating variant, had high levels of cell-associated fluorescence as determined by flow cytometry. Light microscopy indicated that most cell-associated bacteria were ingested. Transmission electron microscopy confirmed the presence of intracellular bacteria, which were contained within membrane-bound phagosomes. A monoclonal antibody specific for the leukocyte integrin polypeptide CD18 partially inhibited attachment of B. bronchiseptica to normal PMN but not to PMN genetically deficient in CD11/CD18 integrins. Higher levels of inhibition occurred when bacteria and normal PMN were co-incubated in the presence of galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, mannose and methyl α-D-mannopyranoside. D-glucose, L-fucose, α-lactose and sialic acid had no inhibitory effect. We conclude that B. bronchiseptica is readily ingested by porcine PMN in the absence of complement and antibody and that internalization is mediated by multiple adhesion mechanisms, including CD18- and carbohydrate-dependent ones.     

Two Crinivirus-specific proteins of Lettuce infectious yellows virus (LIYV), P26 and P9, are self-interacting

Interactions of Lettuce infectious yellows virus (LIYV)-encoded proteins were tested by yeast-two-hybrid (Y2H) assays. LIYV-encoded P34, Hsp70h, P59, CP, CPm, and P26 were tested in all possible pairwise combinations. Interaction was detected only for the P26–P26 combination. P26 self-interaction domains were mapped using a series of N- and C-terminal truncations. Orthologous P26 proteins from the criniviruses Beet pseudoyellows virus (BPYV), Cucurbit yellow stunting disorder virus (CYSDV), and Lettuce chlorosis virus (LCV) were also tested, and each exhibited strong self-interaction but no interaction with orthologous proteins. Two small putative proteins encoded by LIYV RNA2, P5 and P9, were also tested for interactions with the six aforementioned LIYV proteins and each other. No interactions were detected for P5, but P9–P9 self-interaction was detected. P26- and P9-encoding genes are present in all described members of the genus Crinivirus, but are not present in other members of the family Closteroviridae. LIYV P26 has previously been demonstrated to induce a unique LIYV cytopathology, plasmalemma deposits (PLDs), but no role is yet known for P9.     

In vivo detection of lamellocytes in Drosophila melanogaster

Drosophila has recently become a powerful model organism for studies of innate immunity. The cellular elements of innate immunity in Drosophila, the hemocytes, have been characterized by morphological criteria, molecular markers, and cell-type-specific immunological markers. Here we suggest that an MiET1 GFP-reporter element insertion in the untranslated region of a gene (l1-atilla) – expressed in a subset of hemocytes, the lamellocytes – allows in vivo investigations of lamellocyte differentiation and facilitates genetic screens.     

Expression and secretion of pertussis toxin subunit S1 in Bacillus subtilis

Pertussis toxin (PT) is an important virulence determinant of Bordetella pertussis and one of the major protective antigens against whooping cough. The genes coding for PT have recently been cloned, but attempts to express them in Escherichia coli have been unsuccessful. We therefore explored the possibility of expressing these genes in Bacilius subtilis for which efficient vectors are available. The lack of endotoxin in the Gram-positive Bacillus might be an additional advantage for the production of a vaccine component. A DNA fragment coding for S1, one of the subunits of pertussis toxin, was inserted into an alpha-amylase secretion vector and the recombinant plasmid was introduced into B. subtilis. This resulted in high expression of S1, most of which was secreted and therefore found in the culture supernatant. This supernatant had ADP-ribosylating activity similar to that of PT. Western blot with antiserum to B. pertussis holotoxin showed several proteins ranging in size from 28 kDa to 20 kDa reacting in specific manner. About 10% of the protein recognized by the antiserum was of the size expected for native-size S1. The total amount of S1 proteins (full size and truncated) in the culture supernatant was about 100 mg/l. S1 protein made in B. subtilis was partially purified using chromatography with P-cellulose and Blue Sepharose. This preparation was used to immunize rabbits; the immune serum thus obtained recognized subunit S1 of native pertussis toxin.     

The characterization of lectin-binding proteins of Chlamydia trachomatis as glycoproteins

The 18 kDa and 32 kDa lectin binding proteins of Chlamydia trachomatis were characterized as glycoproteins by treatments with glycosidases. The proteins of the serovar L₂ whole cell lysate were separated by SDS-PAGE and transferred to nitrocellulose paper. After treatment with an enzyme, the proteins were reacted with a biotinylated lectin. Each of the endoglycosidases tested affected the binding of the lectin to the protein. PNGase F inhibited the binding of Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), and Ulex europaeus agglutinin I (UEAI) to both the 18 kDa and 32 kDa proteins. Endoglycosidase F and H inhibited the binding of these lectins to the 32 kDa protein completely and to the 18 kDa protein partially. In the exoglycosidase treatments, alpha- -fucosidase prevented binding of only UEAI to the two proteins while beta-galactosidase inhibited the binding of SBA. Mannosidase abolished the binding of all the lectins tested. Neuraminidase had no effect. The proteins isolated by electroelution from the excised gels after SDS-PAGE were digested with an endoglycosidase. PNGase F-treated proteins showed a lower molecular weight mobility in which the lectin binding ability was destroyed. Endo-alpha-N-acetylgalactosaminidase had no effect. The polysaccharide stain of isolated proteins with p-phenylenediamine showed a positive reaction. Radiolabeling with [³H]glucosamine did not reveal the 18 kDa and 32 kDa proteins in autoradiography but [³H]galactose did.     

Systemic allergic reaction to coconut (Cocos nucifera) in 2 subjects with hypersensitivity to tree nut and demonstration of cross-reactivity to legumin-like seed storage proteins: New coconut and walnut food allergens

BACKGROUND: Two patients with tree nut allergy manifested by life-threatening systemic reactions reported the subsequent onset of systemic reactions after the consumption of coconut. OBJECTIVE: Herein, the IgE-binding proteins from coconut are described, and in vitro cross-reactivity with other nuts is investigated. METHODS: The IgE-binding profile of coconut endosperm tissue extract was analyzed by SDS-PAGE followed by immunoblotting. Immunoblot inhibition studies with walnut, almond, peanut, and coconut were performed. RESULTS: Sera IgE from both patients recognized reduced coconut allergens with molecular weights of 35 and 36.5 kd. IgE from 1 patient also bound a 55-kd antigen. Preabsorption of sera with nut extracts suppressed IgE binding to coconut proteins. Preabsorption of sera with coconut caused the disappearance of IgE binding to protein bands at 35 and 36 kd on a reduced immunoblot of walnut protein extract in 1 patient and suppression of IgE binding to a protein at 36 kd in the other patient. CONCLUSION: The reduced coconut protein at 35 kd was previously shown to be immunologically similar to soy glycinin (legumin group of seed storage proteins). The clinical reactivity in these 2 patients is likely due to cross-reacting IgE antibodies primarily directed against walnut, the original clinical allergy reported, and most likely to a walnut legumin-like protein. Coconut allergy in patients with tree nut allergy is rare; these are the first 2 patients ever reported, and therefore there is no general indication to advise patients with tree nut allergy to avoid coconut.     

Nucleotide sequences and characterization of haemolysin genes from Aeromonas hydrophila and Aeromonas sobria

Extracellular haemolysin is thought to be one of the important virulence factors in Aeromonas infection. Two extracellular haemolysin genes (AHH3 and AHH4) from Aeromonas hydrophila strain 28SA, one (AHH5) from A. hydrophila strain AH-1 and one (ASA1) from Aeromonas sobria strain 33 were cloned into cosmid and plasmid vector DNA in Escherichia coli. The nucleotide sequences of the open reading frames of AHH3 and AHH4 are both 1476 basepairs (bp), whereas AHH5 and ASA1 are 1455 and 1467 bp in length, respectively. The deduced amino acid sequences of AHH3, AHH4, AHH5 and the previously reported aerolysin from A. hydrophila showed a significant degree of sequence homology of over 90% each. The amino acid identity of the ASA1 haemolysin and those from A. hydrophila and Aeromonas trota aerolysins ranged from 58–68%. From DNA hybridization analysis using our cloned haemolysin genes as probes, we found that the AHH5 and ASA1 DNA probes hybridized with about 31 and 75% strains of motile Aeromonas species, respectively. The activity of haemolysins of cloned genes were different in medium agar containing various erythrocytes.     

Reproductive characteristics of a precocious vaccine line (Rt3+15) of Eimeria tenella in embryonating chicken eggs

Coccidiosis of chickens, caused by species of Eimeria (Protozoa, Apicomplexa), is an intestinal disease of major economic importance worldwide. In the present study, the reproductive characteristics of a precocious line (designated E. tenella Rt3+15) from Australia were investigated in chicken embryos and the implications of the findings briefly discussed.     

RNAi knock-down of the Litopenaeus vannamei Toll gene (LvToll) significantly increases mortality and reduces bacterial clearance after challenge with Vibrio harveyi

In this study, we used real-time PCR to simultaneously monitor the responses of 12 key genes of the shrimp innate immune system in Litopenaeus vannamei after challenge with Vibrio harveyi. In the proPO activating system, we found that proPO was up-regulated (3.3× control at 36 hpi). The hemolymph clotting genes transglutaminase (TGase) and clotting protein were also up-regulated, as were 5 genes in the antimicrobial peptide system (ALF, Crustin, Lyz, PEN2 and PEN4), with only PEN3 showing no significant changes. In the antioxidant defense system, SOD was slightly elevated while GPx was substantially down-regulated. In the pattern recognition receptor system, at 24 hpi, the Toll gene (LvToll) showed the highest relative increase in expression level of all the investigated genes (15× greater than the sterile seawater control). In the second part of this study, when LvToll was knocked down by RNAi silencing, there was no effect on either survival rates or bacterial number in unchallenged shrimp. There was also no difference in mortality rates between control shrimp and LvToll-silenced shrimp when these two groups were challenged with a viral pathogen (white spot syndrome virus; WSSV). However, when LvToll-silenced shrimp were challenged by V. harveyi, there was a significant increase in mortality and bacterial CFU counts. We note that the increase in bacterial CFU count occurred even though treatment with EGFP dsRNA had the opposite effect of reducing the CFU counts. We conclude that LvToll is an important factor in the shrimp innate immune response to acute V. harveyi infection, but not to WSSV.