Insect-toxic secreted proteins and virulence of the entomopathogenic fungus Beauveria bassiana

Fungal virulence has been mostly associated with cuticle-degrading enzymes that can be regulated depending on nutrient conditions. However, few studies have related fungal virulence to insect-toxic secreted proteins. Here, we describe how the presence of secreted toxic proteins may be linked to conidial virulence, which can be affected by nutrient factors. In this study we evaluated: (1) the virulence of the conidia of four Beauveria bassiana strains (EABb 01/103-Su, EABb 01/12-Su, EABb 01/88-Su and EABb 01/110-Su) grown on three different media (malt extract agar (MA), Rice (Rice), Sabouraud dextrose agar (SDA) and harvested from the cadavers of fungal-infected Galleria mellonella larvae (CAD) and (2) the toxicity of the crude soluble protein extracts (CSPEs) obtained from Adamek’s liquid medium inoculated with these conidia. Conidial suspensions were obtained from the four media, assessed on G. mellonella larvae and used to produce CSPEs that were injected into healthy G. mellonella larvae. The larvae were also injected with conidia obtained from MA and CAD cultures to expose them to in vivo-secreted proteins. For all isolates, the CAD conidia were by far the most virulent, followed by conidia grown on SDA, Rice and MA. The injected CSPEs showed the same toxicity trends as the conidial suspensions. In addition, the outcomes of injection of the in vivo-secreted proteins showed that the toxic proteins secreted in vitro by the EABb 01/110-Su strain are not produced in vivo. However, the other strains produced toxic proteins both in vivo and in vitro, suggesting that these toxic proteins may be virulence factors involved in invertebrate pathogenesis.     

The microRNAs in an Ancient Protist Repress the Variant-Specific Surface Protein Expression by Targeting the Entire Coding Sequence

microRNAs (miRNA) have been detected in the deeply branched protist, Giardia lamblia, and shown to repress expression of the family of variant-specific surface proteins (VSPs), only one of which is expressed in Giardia trophozoite at a given time. Three next-generation sequencing libraries of Giardia Argonaute-associated small RNAs were constructed and analyzed. Analysis of the libraries identified a total of 99 new putative miRNAs with a size primarily in the 26 nt range similar to the size previously predicted by the Giardia Dicer crystal structure and identified by our own studies. Bioinformatic analysis identified multiple putative miRNA target sites in the mRNAs of all 73 VSPs. The effect of miRNA target sites within a defined 3′-region were tested on two vsp mRNAs. All the miRNAs showed partial repression of the corresponding vsp expression and were additive when the targeting sites were separately located. But the combined repression still falls short of 100%. Two other relatively short vsp mRNAs with 15 and 11 putative miRNA target sites identified throughout their ORFs were tested with their corresponding miRNAs. The results indicate that; (1) near 100% repression of vsp mRNA expression can be achieved through the combined action of multiple miRNAs on target sites located throughout the ORF; (2) the miRNA machinery could be instrumental in repressing the expression of vsp genes in Giardia; (3) this is the first time that all the miRNA target sites in the entire ORF of a mRNA have been tested and shown to be functional.     

Interaction of the high-spin Fe atom in the photosystem II reaction center with the quinones QA and QB in purified oxygen-evolving PS II reaction center complex and in PS II particles

EPR signals in the high-spin region were studied at 10 K in photosystem II (PS II) particles and in a purified oxygen-evolving PS II reaction center complex under oxidizing conditions. PS II particles showed EPR peaks at g = 8.0 and 5.6, confirming the recent report by Petrouleas and Diner [(1986) Biochim. Biophys. Acta 849, 264-275]. Addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) or o-phenanthroline shifted the peaks to be closer to g = 6.0 depending on the medium pH. On the other hand, the PS II reaction center complex showed peaks at g = 6.1 and 7.8, and at g = 6.1 and 6.4, in the absence and presence of o-phenanthroline, respectively. All these peaks were found to be decreased by the illumination at 10 K. These results suggest that the high-spin signals are due to Q₄₀₀, Fe(III) atom interacting with the PS II primary electron acceptor quinone Q_A as reported and that the Fe atom also interacts with the secondary acceptor quinone Q_B. This interaction seems to induce the highly asymmetric ligand coordination of the Fe atom and to be affected by DCMU and o-phenanthroline in a somewhat different manner.     

Exploratory analysis of the effect of helminth infection on the immunogenicity and efficacy of the asexual blood-stage malaria vaccine candidate GMZ2

BackgroundHelminths can modulate the host immune response to Plasmodium falciparum and can therefore affect the risk of clinical malaria. We assessed here the effect of helminth infections on both the immunogenicity and efficacy of the GMZ2 malaria vaccine candidate, a recombinant protein consisting of conserved domains of GLURP and MSP3, two asexual blood-stage antigens of P. falciparum. Controlled human malaria infection (CHMI) was used to assess the efficacy of the vaccine.MethodologyIn a randomized, double-blind Phase I clinical trial, fifty, healthy, lifelong malaria-exposed adult volunteers received three doses of GMZ2 adjuvanted with either Cationic Adjuvant Formulation (CAF) 01 or Alhydrogel, or a control vaccine (Rabies) on days (D) 0, D28 and D56, followed by direct venous inoculation (DVI) of 3,200 P. falciparum sporozoites (PfSPZ Challenge) approximately 13 weeks after last vaccination to assess vaccine efficacy. Participants were followed-up on a daily basis with clinical examinations and thick blood smears to monitor P. falciparum parasitemia for 35 days. Malaria was defined as the presence of P. falciparum parasites in the blood associated with at least one symptom that can be associated to malaria over 35 days following DVI of PfSPZ Challenge. Soil-transmitted helminth (STH) infection was assessed by microscopy and by polymerase chain reaction (PCR) on stool, and Schistosoma infection was assessed by microscopy on urine. Participants were considered as infected if positive for any helminth either by PCR and/or microscopy at D0 and/or at D84 (Helm+) and were classified as mono-infection or co-infection. Total vaccine-specific IgG concentrations assessed on D84 were analysed as immunogenicity outcome.Main findingsThe helminth in mono-infection, particularly Schistosoma haematobium and STH were significantly associated with earlier malaria episodes following CHMI, while no association was found in case of coinfection. In further analyses, the anti-GMZ2 IgG concentration on D84 was significantly higher in the S. haematobium-infected and significantly lower in the Strongyloides stercoralis-infected groups, compared to helminth-negative volunteers. Interesting, in the absence of helminth infection, a high anti-GMZ2 IgG concentration on D84 was significantly associated with protection against malaria.ConclusionsOur results suggest that helminth infection may reduce naturally acquired and vaccine-induced protection against malaria. Vaccine-specific antibody concentrations on D84 may be associated with protection in participants with no helminth infection. These results suggest that helminth infection affect malaria vaccine immunogenicity and efficacy in helminth endemic countries.     

Identification of the intermediate host of Gongylonema sp., the etiological agent of the necrotic oropharyngeal disease of the Scops owl (Otus scops)

Since 1997, fledgling Scops owls (Chordata: Strigidae) have been brought to the Brinzal Owl Rescue Centre (Madrid, Spain) with severe lesions in their oral cavities. Lesions consist of the presence of proliferative necrotic material in the oral cavity resulting in white plaques, which can lead to death by starvation. This disease has been detected in owls only within the limits of the city of Madrid. The etiologic agent has been identified as Gongylonema sp. (Nematoda: Spirurida), a nematode genus that includes a coprophagous arthropod as intermediate host in its cycle. The aim of this study was to identify the intermediate host of the parasite. Our work was structured in four component phases: i) Diet study of newborn chicks; ii) trapping arthropods that could be intermediate hosts; iii) molecular detection of the parasite in the selected arthropods: and iv) molecular characterization of the detected parasites by amplifying the cox1 gene. Four male owls were radio-tagged in order to locate their nests and a camera trap was placed to identify the prey brought to the owlets. Secondly, the arthropods present in the hunting areas of the owls were sampled, identified and analyzed by real time PCR (rtPCR). Only oriental cockroaches, B. orientalis (Arthropoda: Blattodea), were positive by rtPCR detection of Gongylonema sp. (66.7%). The nematodes obtained from cockroaches had a 99.8% identity of the cox1 gene with the Gongylonema sp. isolated for the first time in a Scops owl. Furthermore, these sequences only showed an <89% identity with all the other Gongylonema sequences available in the GenBank database. We conclude that the oriental cockroach should be considered as an intermediate host of the etiologic agent of NOD.     

N-methyl-N'-nitro-N-nitrosoguanidine and hydroxylamine induced mutants of the rII region of phage T4

rII mutations of bacteriophage T₄ were induced by in vivo treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NG) and in vitro treatment with hydroxylamine (HA). All the NG induced mutations were mappable to small segments of the rIIA cistron and all except one were also highly revertible by 2-aminopurine (AP) treatment. From these observations, it is concluded that treatment of T₄ with NG induces only transitions and contrary to its effects on E. coli, in T₄, NG does not induce any deletions. Spectra of HA and NG induced mutants of the rIIA cistron were compared. Both mutagens seem to be more effective in inducing mutations nearer the two extremities of this cistron and very few in the middle. This asymmetric effect has been seen to be more pronounced in case of NG than in the case of HA.     

Mutagenesis of Protoplasts and Regeneration of Mycelium in the Mushroom Volvariella volvacea

Regenerating protoplasts were obtained from mycelial culture of the mushroom Volvariella volvacea by the action of the lytic enzyme Novozym 234 in the presence of 0.01 M phosphate buffer (pH 6.0) containing 0.6 M NaCl. Regeneration was found to be poor in liquid medium, but more than 50% regeneration was achieved on solid 2% agar medium overlaid with 0.5% agar. Protoplasts of V. volvacea were found to be highly sensitive to the killing action of both UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine. However, no morphological or auxotrophic mutants could be obtained from protoplasts by chemical mutagenesis. Four types of morphological mutants and one auxotrophic (adenine-negative) mutant were obtained from UV-irradiated protoplasts. The adenine-negative mutant of V. volvacea was found to be stable, not losing auxotrophy on repeated subculture.     

Functional characterization of the octenol receptor neuron on the maxillary palps of the yellow fever mosquito, Aedes aegypti

Background

1-Octen-3-ol (octenol) is a common attractant released by vertebrates which in combination with carbon dioxide (CO₂) attracts hematophagous arthropods including mosquitoes. A receptor neuron contained within basiconic sensilla on the maxillary palps of adult mosquitoes responds selectively to 1-octen-3-ol. Recently, an odorant receptor (AaegOR8) known to occur on the maxillary palps was expressed in a heterologous system and demonstrated to be selectively sensitive to (R)-(−)-1-octen-3-ol, one of two enantiomeric forms. Lesser responses were elicited by stimulation with the (S)-enantiomer and various structural analogs.

Methodology/Principal Findings

Here we characterize the specificity of the octenol receptor neuron in the yellow fever mosquito, Aedes aegypti (L.), in vivo using single cell recordings. The octenol neuron is exquisitely sensitive to (R)-(−)-1-octen-3-ol; comparable responses to (S)-(+)-1-octen-3-ol were elicited only at stimulus doses over 100× that required for the (R)-enantiomer. An intermediate response closer to that elicited by the (R)-(−)-enantiomer was elicited by racemic 1-octen-3-ol. Small structural changes in (R)-(−)-1-octen-3-ol resulted in large decreases in responses. Increases in spike activity were also elicited in the octenol neuron by 2-undecanone, a known repellent; other repellents (DEET, IR3535 and picaridin) were inactive.

Conclusions/Significance

The results of our electrophysiological studies of the octenol receptor neuron in vivo approximates results of a previous study of the octenol receptor (AaegOR8 with its obligate partner Aaeg\ORco) expressed heterologously in Xenopus oocytes. By comparison of our current results with those of the heterologous expression study, we conclude that specificity of the octenol receptor neuron can be explained largely by characteristics of the OR alone without other associated proteins present in vivo. Our findings show that repellents may have specific stimulatory effects on receptor neurons and support the notion of repellents as modulators of mosquito odorant receptor activity.     

Vegetative incompatibility in the ash dieback pathogen Hymenoscyphus pseudoalbidus and its ecological implications

Pairings were carried out between isolates of the ash dieback pathogen Hymenoscyphus pseudoalbidus (Chalara fraxinea) to determine whether vegetative incompatibility (mycelial self–nonself recognition) reactions could be discriminated. On malt agar (MA) and ash sapwood agar (ASA) distinct compatible and incompatible reactions were observed. Compatible (C- or c-) reactions were characterised by full or partial colony intermingling along the junction line. Incompatible (G- or g-) reactions were characterised by a gap ca. 1–3 mm wide along the junction line. On MA a distinct narrow brown line or L-reaction was observed with some gap reactions, often comprising reticulate mycelium and conidia-producing phialides. When eleven isolates from a dieback site at Lower Wood, Norfolk were paired on ASA 53 of 54 reactions between different isolates gave incompatible reactions. A similar result was obtained with smaller samples from two further sites in Norfolk and Kent. This indicates that for local populations in the UK most genetic individuals of H. pseudoalbidus are likely to be vegetatively incompatible. The implications for the ecology and genetics of H. pseudoalbidus are discussed.     

Coal Depyritization by the Thermophilic Archaeon Metallosphaera sedula

The kinetics of pyrite oxidation by Metallosphaera sedula were investigated with mineral pyrite and two coals with moderate (Pittsburgh no. 8) and high (New Brunswick, Canada) pyritic sulfur content. M. sedula oxidized mineral pyrite at a greater rate than did another thermophile, Acidianus brierleyi, or a mesophile, Thiobacillus ferrooxidans. Maximum rates of coal depyritization were also greater with M. sedula, although the magnitude of biological stimulation above abiotic rates was notably less than with mineral pyrite. Coal depyritization appears to be limited by the oxidation of pyrite with ferric ions and not by the rate of biotic oxidation of ferrous iron, as evidenced by the maintenance of a high ratio of ferric to ferrous iron in solution by M. sedula. Significant precipitation of hydronium jarosite at elevated temperature occurred only with New Brunswick coal.     

Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a “probe dropout” manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.     

Neither castration nor steroid-replacement change the apparent molecular size of FSH in the sheep pituitary

Gonadal steroids alter the apparent molecular size of intrapituitary Follicle-Stimulating Hormone (FSH) in rats and monkeys as well as increase the percentage of acidic FSH isohormones in sheep. Hence, we hypothesized that the molecular size of ovine (o) FSH would be increased by gonadal steroids. Extracts of pituitaries from rams and wethers, as well as, from wethers which had been implanted with dihydrotestosterone (DHT), 17β-estradiol (E₂) or both steroids (n=4–6 per treatment group) were subjected to analytical gel permeation chromatography using Sephadex G-100 Superfine. FSH concentrations in chromatographic fractions were determined by radioimmunoassays. Although FSH in pituitaries of non-implanted wethers eluted slightly earlier (i.e. larger) than FSH in pituitaries from E₂-implanted wethers as evaluated by distribution coefficients (K_ds) during chromatography (P<0.05), gonadal steroids did not consistently increase K_ds but tended to decrease them. When K_ds were extrapolated to apparent molecular weights using a series of standard proteins (bovine serum albumin (bSA), ovalbumin (OA), carbonic anhydrase (CA) and cytochrome c (CC)) that were included in each chromatogram, the differences between treatment groups were not statistically significant (P>0.05). Thus, in contrast to rats and monkeys, neither castration nor steroid-replacement appears to alter the molecular size of FSH in the sheep pituitary as evaluated by analytical gel permeation chromatography.     

Role of self carriers in the immune response and tolerance. III. B cell tolerance induced by hapten-modified-self involves both active T cell-mediated suppression and direct blockade.

The induction of B cell unresponsiveness with hapten-modified syngeneic murine lymphoid cells (hapten-modified self, HMS) can be achieved in vivo and in vitro. Tolerance in vivo in mice required a latent period of 3 to 4 days. Moreover, B cell unresponsiveness could not be induced by HMS in athymic nude mice, although their nu/+ littermates were rendered hyporesponsive by HMS. Pretreatment of normal mice with cyclophosphamide (cyclo) prevented their susceptibility to tolerance induction by haptenated lymphoid cells. Nude mice became sensitive to HMS-induced suppression if they were first reconstituted with spleen cells from normal (but not cyclo-treated) donors.Interestingly, labeling of H-2 antigens was not necessary for tolerance induction by HMS since haptenated teratoma cells (lacking H-2) were tolerogenic in normal recipients.In contrast, suppression of the in vitro response to haptenated flagellin occurred equally well with nude, nu/+ and anti-Ly 2 + C-treated spleen cells. These data suggest that cyclo-sensitive modified self-reactive (T) cells may regulate the immune response and mediate tolerance to HMS in vivo. However, the in vitro “blockade” of B cell reactivity may be directly mediated on hapten-specific PFC precursors.     

Combining various strategies to increase the coverage of the plant cell wall glycoproteome

Glycoproteomics recently became a very active field, mostly in mammals. The first part of this paper consists of a mini-review on the strategies used in glycoproteomics, namely methods for enrichment in glycoproteins and mass spectrometry (MS) techniques currently used. In a second part, these strategies are applied to the cell wall glycoproteome of etiolated hypocotyls of Arabidopsis thaliana, showing their complementarity. Several sub-glycoproteomes were obtained by: (i) affinity chromatography on concanavaline A (ConA) and analysis of glycoproteins by MALDI-TOF MS; (ii) multidimensional lectin chromatography (using AIL, PNA, ConA and WGA lectins) and subsequent identification of glycoproteins by MALDI-TOF MS and LC-MS/MS; (iii) boronic acid chromatography followed by identification of glycoproteins by MALDI-TOF MS. Altogether, 127 glycoproteins were identified. Most glycoproteins were found to be putative N-glycoproteins and N-glycopeptides were predicted from MS data using the ProTerNyc bioinformatics software.     

Animal-based measures on fattening heavy pigs at the slaughterhouse and the association with animal welfare at the farm level: a preliminary study

Monitoring animal welfare (AW) in pig farms requires both proper indicators and a feasible approach. Animal-based measures (ABMs) are well-established AW indicators. Furthermore, AW screening at the slaughterhouses could be useful for identifying problems on farm. The aim of this study was to evaluate ABMs at the slaughterhouse and, when possible, to compare these ABMs with those collected on the farm. The study was carried out in northern Italy in a commercial abattoir and in a sample of farms. Animal-based measures were recorded on pigs from 62 batches of 54 farms, during ante-mortem (n=10 085 pigs) and post-mortem (n=7952 pigs) inspections. Sixteen of 54 farms were selected to compare ABMs collected at the slaughterhouse with ABMs collected on the farm. Overall, 2295 pigs (mean pigs examined per farm 119±45) were inspected at the slaughterhouse (group S) and 420 pigs (mean pigs per farm 26±5) on the farm (group F). Non-animal-based measures were also collected at the 16 farms. Differences between groups S and F, at the animal level, were assessed by a two-tailed paired Wilcoxon–Mann–Whitney test. Differences at the site of observation level (farm and slaughterhouse) were assessed by Fisher’s exact test using a hierarchical log-linear modelling for contingency tables. The most frequent ABMs at the slaughterhouse were manure on the body (47.7%), followed by dermatitis (28.0%), white spot (25.4%) and bursitis (24.7%). Recording ABMs at the slaughterhouse and on the farm usually yielded similar results; however, there were some exceptions. In particular, significant differences were found for non-uniformity of size (P<0.05) and dermatitis (P<0.001), which were higher at the slaughterhouse than on the farm. Results of log-linear modelling underlined the effect of the farm of origin on the percentage of pigs with bursitis, manure on the body and ear injuries that were observed at the slaughterhouse. In group S, significant associations between manure on the body and insufficient presence of clean and dry areas in the corresponding farm were found (P<0.05). Although these results should be interpreted with care due to the limited sample of farms, the slaughterhouse could be a feasible site of observation of ABMs, which could then be integrated in monitoring of AW on farm. Considering the number of slaughtered batches per farm, it would be possible to repeat assessments several times throughout the year for each farm, which could help provide an index for the continuous monitoring of AW.     

Microbial diversity from the continental shelf regions of the Eastern Arabian Sea: A metagenomic approach

The marine microbiome is a complex and least-understood habitat, which play a significant role in global biogeochemical cycles. The present study reported the culture-independent assessment of microbial diversity from the Arabian Sea (AS) sediments (from Gujarat to Malabar; at 30 m depth) by using metagenome sequence analysis. Our results elucidated that bacterial communities in the Malabar coastal region are highly diverse than the Gujarat coast. Moreover, Statistical analysis (Spearman rank correlation) showed a significant correlation co-efficient value (r = P < 0.001) between microbial communities and physicochemical parameters (salinity and dissolved oxygen) in the water column. A total of 39 bacterial phyla were recorded from the eastern side of AS, of which six phyla Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, Firmicutes, and Planctomycetes were found to be the most dominant group. The most dominant genus from Valapad region (Malabar Coast) was found to be Halomonas sp., while other regions were dominated with Psychrobacter pulmonis. The subsequent Principal Coordinate Analysis (PCoA) showed 99.53% variance, which suggests that, highly distinct microbial communities at Valapad (Malabar Coast) sampling location than other sites. Moreover, the microbial metabolic activity analysis revealed the important functions of microbial communities in the AS are hydrocarbon degradation, polymer degradation, nutrient oxidation and sulphate reduction (biodegradation process). Further extended studies are needed to be carried out for better understanding the functional diversity of microbial communities from the marine sediments.     

Syntheses of some purine nucleosides by the fusion method,by the condensation of acetylated chlorides,and from 1-acetates in the presence of titanium tetrachloride: a comparison

9-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-6-benzamidopurine (9) and 6-benzamido-9-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)purine (11) have been prepared by three synthetic routes: (a) the fusion procedure, (b) direct condensation of 6-benzamido(chloromercuri)purine with the acetylated chloride, or (c) with the chloride formed in situ from the 1-acetate in the presence of titanium tetrachloride. The results obtained are briefly discussed; the direct condensation of the mercuri salt with chlorides proved to be the most convenient.Whereas, in the condensation with acetylated chlorides, only products having the β-d anomeric configuration were isolated, the chloride protected with non-participating groups (benzyl) afforded both anomers. The removal of the benzyl groups should be preceded by hydrolytic cleavage of the benzamido group. A simple procedure for fractionation, on small columns of silica gel, of reaction mixtures obtained in the fusion reactions is described.