Isolation and characterization of a Cotesia rubecula bracovirus gene expressed in the lepidopteran Pieris rapae

Polydnaviruses are endogenous particles that are crucial for the survival of endoparasitoid wasps, providing active suppression of the immune function of the lepidopteran host in which wasp larvae develop. The Cotesia rubecula bracovirus (CrBV) is unique in that only four gene products are detected in larval host (Pieris rapae) tissues and expression of CrBV genes is transient, occurring between 4 and 12 h post-parasitization. Two of the four genes, CrV1 and CrV3, have been characterized. CrV1 is a secreted glycoprotein that has been implicated in depolymerization of the actin cytoskeleton of host haemocytes, leading to haemocyte inactivation; CrV3 is a multimeric C-type lectin that shares homology with insect immune lectins. Here, a third CrBV-specific gene is described, CrV2, which is expressed in larval P. rapae tissues. CrV2, which is transcribed in haemocytes and fat body cells, has an ORF of 963 bp that produces a glycoprotein of approximately 40 kDa. CrV2 is secreted into haemolymph and appears to be internalized by host haemocytes. CrV2 has a coiled-coil region predicted at its C-terminus, which may be involved in the formation of putative CrV2 trimers that are detected in haemolymph of parasitized host larvae.

Lectin-induced haemocyte inactivation in insects

Most multimeric lectins are adhesion molecules, promoting attachment and spreading on surface glycodeterminants. In addition, some lectins have counter-adhesion properties, detaching already spread cells which then acquire round or spindle-formed cell shapes. Since lectin-mediated adhesion and detachment is observed in haemocyte-like Drosophila cells, which have haemomucin as the major lectin-binding glycoprotein, the two opposite cell behaviours may be the result of lectin-mediated receptor rearrangements on the cell surface. To investigate oligomeric lectins as a possible extracellular driving force affecting cell shape changes, we examined lectin-mediated reactions in lepidopteran haemocytes after cytochalasin D-treatment and observed that while cell-spreading was dependent on F-actin, lectin-uptake was less dependent on F-actin. We propose a model of cell shape changes involving a dynamic balance between adhesion and uptake reactions. (C) 2004 Elsevier Ltd. All rights reserved.

Are insect immune suppressors driving cellular uptake reactions?

Many insect parasitoids that deposit their eggs inside immature stages of other insect species inactivate the cellular host defence to protect the growing embryo from encapsulation. Suppression of encapsulation by polydnavirus-encoded immune-suppressors correlates with specific alterations in hemocytes, mainly cytoskeletal rearrangements and actin-cytoskeleton breakdown. We have previously shown that the Cotesia rubecula polydnavirus gene product CrV1 causes immune suppression when injected into the host hemocoel. CrV1 is taken up by hemocytes although no receptors have been found to bind the protein. Instead CrV1 uptake depends on dimer formation, which is required for interacting with lipophorin, suggesting a CrV1-lipophorin complex internalisation by hemocytes. Since treatment of hemocytes with oligomeric lectins and cytochalasin D can mimic the effects of CrV1, we propose that some dimeric and oligomeric adhesion molecules are able to cross-link receptors on the cell surface and depolymerise actin by leverage-mediated clearance reactions in the hemolymph.

A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy

The gastrointestinal tracts of multi-cellular blood-feeding parasites are targets for vaccines and drugs. Recently, recombinant vaccines that interrupt the digestion of blood in the hookworm gut have shown efficacy, so we explored the intestinal transcriptomes of the human and canine hookworms, Necator americanus and Ancylostoma caninum, respectively. We used Laser Microdissection Microscopy to dissect gut tissue from the parasites, extracted the RNA and generated cDNA libraries. A total of 480 expressed sequence tags were sequenced from each library and assembled into contigs, accounting for 268 N. americanus genes and 276 A. caninum genes. Only 17% of N. americanus and 36% of A. caninum contigs were assigned Gene Ontology classifications. Twenty-six (9.8%) N. americanus and 18 (6.5%) A. caninum contigs did not have homologues in any databases including dbEST-of these novel clones, seven N. americanus and three A. caninum contigs had Open Reading Frames with predicted secretory signal peptides. The most abundant transcripts corresponded to mRNAs encoding cholesterol-and fatty acid-binding proteins, C-type lectins, Activation-Associated Secretory Proteins, and proteases of different mechanistic classes, particularly astacin-like metallopeptidases. Expressed sequence tags corresponding to known and potential recombinant vaccines were identified and these included homologues of proteases, anti-clotting factors, defensins and integral membrane proteins involved in cell adhesion. (c) 2006 Australian Society for Parasitology Inc Published by Elsevier Ltd. All fights reserved.

Expression of human DEC-205 (CD205) multilectin receptor on leukocytes

DEC-205 (CD205) belongs to the macrophage mannose receptor family of C-type lectin endocytic receptors and behaves as an antigen uptake/processing receptor for dendritic cells (DC). To investigate DEC-205 tissue distribution in human leukocytes, we generated a series of anti-human DEC-205 monoclonal antibodies (MMRI-5, 6 and 7), which recognized epitopes within the C-type lectin-like domains 1 and 2, and the MMRI-7 immunoprecipitated a single similar to 200 kDa band, identified as DEC-205 by mass spectrometry. MMRI-7 and another DEC-205 mAb (MG38), which recognized the epitope within the DEC-205 cysteine-rich and fibronectin type II domain, were used to examine DEC-205 expression by human leukocytes. Unlike mouse DEC-205, which is reported to have predominant expression on DC, human DEC-205 was detected by flow cytometry at relatively high levels on myeloid blood DC and monocytes, at moderate levels on B lymphocytes and at low levels on NK cells, plasmacytoid blood DC and T lymphocytes. MMRI-7 F(ab')(2) also labeled monocytes, B lymphocytes and NK cells similarly excluding reactivity due to non-specific binding of the mAb to Fc gamma R. Tonsil mononuclear cells showed a similar distribution of DEC-205 staining on the leukocytes. DEC-205-specific semiquantitative immunoprecipitation/western blot and quantitative reverse transcriptase-PCR analysis established that these leukocyte populations expressed DEC-205 protein and the cognate mRNA. Thus, human DEC-205 is expressed on more leukocyte populations than that were previously assumed based on mouse DEC-205 tissue localization studies. The broader DEC-205 tissue expression in man is relevant to clinical DC targeting strategies and DEC-205 functional studies.

Determination of Coniothyrium minitans conidial and germling lectin avidity by flow cytometry and digital microscopy

The avidity of conidia and 48-h-old germlings of Coniothyrium minitans for FITC-conjugated lectins was characterised by flow cytometry and digital microscopy. Six isolates of C. minitans representing three morphological types were compared. Binding of Con A, SBA and WGA by conidial populations varied markedly in extent and pattern between isolates, however, with increasing culture age, conidia from all isolates demonstrated a significant reduction in lectin avidity. Germling isolates bound significantly different amounts of lectins and lectin binding differed significantly with locality. Spore walls of all germlings from all isolates bound more ConA compared with hyphal apices and mature hyphal walls. In contrast, hyphal apices of the majority of germling isolates, readily bound SBA and mature hyphal walls of germling isolates bound more WGA than other regions of the germlings. Such differential lectin binding by conidia and germlings may influence their specific surface interactions and adherence characteristics.

Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and flourescence evaluation

Aims: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. Methods and Results: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K d and relative Bmax values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, Kd and relative B max, which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. Conclusions: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. Significance and Impact of the Study: The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

Characterization of FITC-conjugated lectin binding to Candida albicans

Avidity of yeast and hyphal forms of Candida albicans for FITC-conjugated lectins was determined by flow cytometry and digital microscopy. Yeast phase cells bound Con A, a lectin with marked affinity for mannose, irrespective of growth phase, yet demonstrated little avidity for WGA and SBA. Yeast phase cell avidity for mannose-specific lectins was characterized through determination of FITC-conjugated Con A, LcH, PSA and GNA binding and subsequent calculation of Bmax, EC50 and Hn values. Such an approach, through comparison among FITC-conjugated lectins of differing specific activities, furnishes further insight into exposed outer cell wall mannose moieties. The rank order of lectin affinity as defined by EC50 values was GNA > Con A > LcH > PSA. Values for Hn suggest that lectins predominantly bind to a single receptor class, the relative abundance of which as defined by Bmax values was PSA > GNA > Con A > LcH. Hyphal surfaces in common with yeast phase cells demonstrated marked avidity for FITC-Con A, however, fluorescence of Candida morphological forms differed significantly, indicative of varying outer cell wall mannose exposure.

Acanthamoebal surface properties and the modulation of phagocytosis

The surface nature of Acanthamoeba trophozoites and cysts was investigated with respect to cell surface charge, hydrophobicity and surface carbohydrate composition. Particulate microelectrophoresis revealed a marked negative charge for both morphological forms, though less for cyst surfaces. Hydrophobicity was determined by adhesion to n-hexadecane and indicated a relatively low hydrophobic nature of both forms, though less so for cysts. Surface carbohydrate composition was studied by the use of fluorescent lectins and flow cytometry, using a ligand-receptor approach for further in depth analysis of binding of particular lectins. These studies showed trophozoite and cyst surfaces to be rich in N-acetylglucosamine, N-acteylneuraminic acid, mannose and glucose, with the addition of N-acetylgalactosamine on cysts. The importance of such surface properties was investigated with respect to phagocytosis of polystyrene latex microspheres, of different surface types and size. Investigations into the optimum conditions of uptake of beads indicated a preference for a medium devoid of nutrients, such as saline, though temperature was not a factor. An amoebal predilection for beads of lower charge and greater hydrophobicity was demonstrated. Furthermore, a preference for the largest bead size used (2.0 m) was observed. The influence of either Con A or mannose or glucose on bead association was apparently limited. The fate of foreign DNA ingested by Acanthamoeba appeared to indicate that such DNA was destroyed, as it could not be detected following extraction procedures and PCR amplification.

Cell surface analysis of the basidiomycete yeast cryptococcus neoformans

Cell surface properties of the basidiomycete yeast Cryptococcus neoformans were investigated with a combination of novel and well proven approaches. Non-specific cell adhesion forces, as well as exposed carbohydrate and protein moieties potentially associated with specific cellular interaction, were analysed. Experimentation and analysis employed cryptococcal cells of different strains, capsular status and culture age. Investigation of cellular charge by particulate microelectrophoresis revealed encapsulated yeast forms of C. neoformans manifest a distinctive negative charge regardless of the age of cells involved; in turn, the neutral charge of acapsulate yeasts confirmed that the polysaccharide capsule, and not the cell wall, was responsible for this occurrence. Hydrophobicity was measured by MATH and HICH techniques, as well as by the attachment of polystyrene microspheres. All three techniques, where applicable, found C. neoformans yeast to be consistently hydrophilic; this state varied little regardless of strain and culture age. Cell surface carbohydrates and protein were investigated with novel fluorescent tagging protocols, flow cytometry and confocal microscopy. Cell surface carbohydrate was identified by controlled oxidation in association with biotin hydrazide and fluorescein-streptavidin tagging. Marked amounts of carbohydrate were measured and observed on the cell wall surface of cryptococcal yeasts. Furthermore, tagging of carbohydrates with selective fluorescent lectins supported the identification, measurement and observation of substantial amounts of mannose, glucose and N-acetyl-glucosamine. Cryptococcal cell surface protein was identified using sulfo-NHS-biotin with fluorescein-streptavidin, and then readily quantified by flow cytometry. Confocal imaging of surface exposed carbohydrate and protein revealed common localised areas of vivid fluorescence associated with buds, bud scars and nascent daughter cells. Carbohydrate and protein fluorescence often varied between strains, culture age and capsule status of cells examined. Finally, extension of protein tagging techniques resulted in the isolation and extraction of two biotinylated proteins from the yeast cell wall surface of an acapsulate strain of C.neoformans.

The control of mitosis in mammalian tissues

The question of which factors are central in determining whether a cell will undertake a new round of mitosis or will decycle has been examined in the isolated thymic lymphocyte model. Such cell populations possess both in vivo and in vitro a subpopulation of quiescent lymphoblasts which may be induced to reinitiate their mitotic programme. In the intact animal the major determinant of proliferative activity is the plasma ionised calcium concentration. However it has been established in culture that a variety of hormones, ions, cyclic nucleotides, plant lectins and ionophores may like calcium elicit a mitogenic response. These agents do not appear however to initiate DNA synthesis in an identical fashion. Rather there are two distinct intracellular mitogenic axes. The first axis includes a number of adenylate cyclase stimulants, cyclic AMP, phosphodiesterase inhibitors and magnesium ions. It was found that all these mitogens required extracellular magnesium ions to exhibit their stimulatory capacity. This dichotomy in mitogenic activity was further emphasised by the observation that these mitogens are all inhibited by testosterone, whilst the magnesium-independent mitogens were insensitive to this androgen. Indeed this second group of stimulatory factors required the presence of calcium ions in the extracellular milieu for activity, and were, in contrast to the magnesium-dependent mitogens inhibited by the presence of oestradiol in the culture. By examining the interrelationships between these various mitogens and inhibitors it has been possible to propose a mechanism to describe the activation process in the thymocyte. Studies of the metabolism of cyclic nucleotides, membrane potential and transmembrane ion fluxes indicate that there may be a complex relationship between membrane fluidity, ion balance and cyclic nucleotide levels which may individually or in concert promote the initiation of DNA synthesis. A number of possible mechanisms are discussed to account for these observations.

The effect of R-plasmid RP1 on the properties of Proteus mirabilis

A clinical isolate of Proteus mirabilis containing R-plasmid RP1 (R+ cells), grown in both iron- and carbon- limited chemically defined media in mixed culture with plasmid-free (R- cells), did not disappear as expected, due to adherence of R+ cells to the wall of the chemostat vessel. Plasmid RP1 promoted adherence to glass and to medical prostheses. The hydrophobicity and surface charge of R+ cells were different from those of R- cells and both factors may contribute to the adherence of R+ cells to surfaces. The mode of cultivation of the cells, whether batch or continuous culture, were also found to affect the result. Antibodies raised against homologous cells increased the surface hydrophobicity of both R+ and R- cells and eliminated the differences between them. Results for surface hydrophobicity varied with the method used for measuring it. R+ cells were more sensitive than R- cells to tbe bacteridical action of normal serum and whole blood and to phagocytosis as measured by chemiluminescence. No clear differences were revealed in the protein antigens of R+ and R- cells by both SDS PAGE gels and immunoblots reacted with homologous antibodies. However, lectins revealed differences in the sugars exposed on the cell surfaces. Chemical analysis of R&43 and R- cells also revealed differences in the content of 2-keto-3-deoxy-D-manno-2-octulosonate, lipopolysaccharide and total fatty acids, when cells were grown in media containing added iron; however, no qualitative differences in the lipopolysaccharide were found. Removal of iron from the medium was found to have considerable effects on the chemical structure of R+ cells but not of R- ones. Adhesion to prostheses and to leucocytes is discussed in the light of the results and the clinical relevance outlined with respect to the initiation of infection and the association of virulence with antibiotic resistance.

Enterotoxigenic Escherichia coli infection of pigs: expression, extraction, purification and the adhesive properties of the K88 fimbrial adhesin

The ability of Escherichia coli to express the K88 fimbrial adhesin was satisfactorily indicated by the combined techniques of ELISA, haemagglutination and latex agglutination. Detection of expression by electron microscopy and the ability to metabolize raffinose were unsuitable. Quantitative expression of the K88 adhesin was determined by ELISA. Expression was found to vary according to the E.coli strain examined, media type and form. In general it was found that the total amount was greater, while the amount/cfu was less on agar than in broth cultures. Expression of the K88 adhesin during unshaken batch culture was related to the growth rate and was maximal during late logarithmic to early stationary phase. A combination of heat extraction, ammonium sulphate and isoelectric precipitation was found suitable for both large and small scale preparation of purified K88ab adhesin. Extraction of the K88 adhesin was sensitive to pH and it was postulated that this may affect the site of colonisation of by ETEC in vivo. Results of haemagglutination experiments were consistent with the hypothesis that the K88 receptor present on erythrocytes is composed of two elements, one responsible for the binding of K88ab and K88ac and a second responsible for the binding of the K88ad adhesin. Comparison of the haemagglutinating properties of cell-free and cell-bound K88 adhesin revealed some differences probably indicating a minor conformational change in the K88 adhesin on its isolation. The K88ab adhesin was found to bind to erythrocytes over a wide pH range (PH 4-9) and was inhibited by αK88ab and αK88b antisera. Inhibition of haemagglutination was noted with crude heparin, mannan and porcine gastric mucin, chondrosine and several hexosamines, glucosamine in particular. The most potent inhibitor of haemagglutination was n-dodecyl-β-D-glucopyranoside, one of a series of glucosides found to have inhibitory properties. Correlation between hydrophobicity of glucosides tested and degree of inhibition observed suggested hydrophobic forces were important in the interaction of the K88 adhesin with its receptor. The results of Scatchard and Hill plots indicated that binding of the K88ab adhesin to porcine enterocytes in the majority of cases is a two-step, three component system. The first K88 receptor (or site) had a K2. of 1.59x1014M-1 and a minimum of 4.3x104 sites/enterocyte. The second receptor (or site) had a K2 of 4.2x1012M-1 with a calculated 1.75x105 sites/enterocyte. Attempts to inhibit binding of cell-free K88 adhesin to porcine enterocytes by lectins were unsuccessful. However, several carbohydrates including trehalose, lactulose, galactose 1→4 mannopyranoside, chondrosine, galactosamine, stachyose and mannan were inhibitory. The most potent inhibitor was found to be porcine gastric mucin. Inhibition observed with n-octyl-α-D-glucopyranose was difficult to interpret in isolation because of interference with the assay, however, it agreed with the results of haemagglutination inhibition experiments.

FITC-lectin avidity of Cryptococcus neoformans cell wall and capsular components

Flow cytometry and confocal microscopy were used to quantify and visualize FITC-lectin binding to cell-surface carbohydrate ligands of log and stationary phase acapsular and capsular Cryptococcus neoformans strains. Cell populations demonstrated marked avidity for terminal a-linked mannose and glucose specific FITC-Con A, mannose specific FITC-GNL, as well as N-acetylglucosamine specific FITC-WGA. Exposure to other FITC-lectins specific for mannose, fucose and N-acetylgalactosamine resulted in little cell-surface fluorescence. The nature of cell-surface carbohydrates was investigated further by measurement of the fluorescence from surfaces of log and stationary phase cell populations after exposing them to increasing concentrations of FITC-Con A and FITC-WGA. Cell fluorescence increased significantly with small increases in FITC-Con A and FITC-WGA concentrations attaining reproducible maxima. Measurements of this nature supported calculation of the lectin binding determinants EC 50, Hn, Fmax and relative Bmax values. EC50 values indicated that the yeast-cell surfaces had greatest affinity for FITC-WGA, however, relative Bmax values indicated that greater numbers of Con A binding sites were present on these same cell surfaces. Hn values suggested a co-operative lectin-carbohydrate ligand interaction. Imaging of FITC-Con A and FITC-WGA cell-surface fluorescence by confocal microscopy demonstrated marked localization of both lectins to cell surfaces associated with cell division and maturation, indicative of dynamic carbohydrate ligand exposure and masking. Some fluorescence was associated with entrapment of FITC-Con A by capsular components, but FITC-Con A and FITC-WGA readily penetrated the capsule matrix to bind to the same cell surfaces labelled in acapsular cells.

Coral Thermal Tolerance: Tuning Gene Expression to Resist Thermal Stress

The acclimatization capacity of corals is a critical consideration in the persistence of coral reefs under stresses imposed by global climate change. The stress history of corals plays a role in subsequent response to heat stress, but the transcriptomic changes associated with these plastic changes have not been previously explored. In order to identify host transcriptomic changes associated with acquired thermal tolerance in the scleractinian coralAcropora millepora, corals preconditioned to a sub-lethal temperature of 3°C below bleaching threshold temperature were compared to both non-preconditioned corals and untreated controls using a cDNA microarray platform. After eight days of hyperthermal challenge, conditions under which non-preconditioned corals bleached and preconditioned corals (thermal-tolerant) maintained Symbiodinium density, a clear differentiation in the transcriptional profiles was revealed among the condition examined. Among these changes, nine differentially expressed genes separated preconditioned corals from non-preconditioned corals, with 42 genes differentially expressed between control and preconditioned treatments, and 70 genes between non-preconditioned corals and controls. Differentially expressed genes included components of an apoptotic signaling cascade, which suggest the inhibition of apoptosis in preconditioned corals. Additionally, lectins and genes involved in response to oxidative stress were also detected. One dominant pattern was the apparent tuning of gene expression observed between preconditioned and non-preconditioned treatments; that is, differences in expression magnitude were more apparent than differences in the identity of genes differentially expressed. Our work revealed a transcriptomic signature underlying the tolerance associated with coral thermal history, and suggests that understanding the molecular mechanisms behind physiological acclimatization would be critical for the modeling of reefs in impending climate change scenarios.