A phylogenomic approach to reconstructing the diversification of serine proteases in fungi

Using a phylogenomic approach with 10 fungi of very different virulence and habitat, we determined that there was substantial diversification of subtilase-type proteases early in ascomycete history (with subsequent loss in many lineages) but with no comparable diversification of trypsins. Patterns of intron loss and the degree of divergence between paralogues demonstrated that the proliferation of proteinase K subtilases and subtilisin type subtilases seen in pathogenic ascomycetes (Metarhizium anisopliae, Magnaporthe grisea, Fusarium graminearum) occurred after the basidiomycete/ascomycete split but predated radiation of ascomycete lineages. This suggests that the early ascomycetes had a lifestyle that selected for multiple proteases, whereas the current disparity in gene numbers between ascomycete lineages results from retention of genes in at least some pathogens that have been lost in other lineages (yeasts, Aspergillus nidulans, Neurospora crassa). A similar prevailing trend towards lineage specific gene loss of trypsins in saprophytes and some pathogens suggests that their phylogenetic breadth will have been much wider in early fungi than currently.     

The multivesicular body-localized GTPase ARFA1b/1c is important for callose deposition and ROR2 syntaxin-dependent preinvasive basal defense in barley

Host cell vesicle traffic is essential for the interplay between plants and microbes. ADP-ribosylation factor (ARF) GTPases are required for vesicle budding, and we studied the role of these enzymes to identify important vesicle transport pathways in the plant-powdery mildew interaction. A combination of transient-induced gene silencing and transient expression of inactive forms of ARF GTPases provided evidence that barley (Hordeum vulgare) ARFA1b/1c function is important for preinvasive penetration resistance against powdery mildew, manifested by formation of a cell wall apposition, named a papilla. Mutant studies indicated that the plasma membrane-localized REQUIRED FOR MLO-SPECIFIED RESISTANCE2 (ROR2) syntaxin, also important for penetration resistance, and ARFA1b/1c function in the same vesicle transport pathway. This was substantiated by a requirement of ARFA1b/1c for ROR2 accumulation in the papilla. ARFA1b/1c is localized to multivesicular bodies, providing a functional link between ROR2 and these organelles in penetration resistance. During Blumeria graminis f sp hordei penetration attempts, ARFA1b/1c-positive multivesicular bodies assemble near the penetration site hours prior to the earliest detection of callose in papillae. Moreover, we showed that ARFA1b/1c is required for callose deposition in papillae and that the papilla structure is established independently of ARFA1b/1c. This raises the possibility that callose is loaded into papillae via multivesicular bodies, rather than being synthesized directly into this cell wall apposition.     

Myristic acid increases delta6-desaturase activity in cultured rat hepatocytes

In order to study the effects of saturated fatty acids on delta6-desaturase activity, rat hepatocytes in primary culture were incubated with lauric (C12:0), myristic (C14:0) or palmitic (C16:0) acids. After optimization, the standard in vitro conditions for the measurement of delta6-desaturase activity were as follows: 60 micromol x L(-1) alpha-linolenic acid (C18:3n-3), reaction time of 20 min and protein content of 0.4 mg. Data showed that cell treatment with 0.5 mmol x L(-1) myristic acid during 43 h specifically increased delta6-desaturase activity. This improvement, reproducible for three substrates of delta6-desaturase, i.e. oleic acid (C18:1n-9), linoleic acid (C18:2n-6) and alpha-linoleic acid (C18:3n-3) was dose-dependent in the range 0.1-0.5 mmol x L(-1) myristic acid concentration.     

Identification of thermostable β-xylosidase activities produced by <Emphasis Type="Italic">Aspergillus brasiliensis</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicus produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable β-xylosidases. The β-xylosidase activities of the A. brasiliensis and A. niger strains had similar temperature and pH optima at 75°C and pH 5 and retained 62% and 99%, respectively, of these activities over 1 h at 60°C. At 75°C, these values were 38 and 44%, respectively. Whereas A. niger is a well known enzyme producer, this is the first report of xylanase and thermostable β-xylosidase production from the newly identified, non-ochratoxin-producing species A. brasiliensis.     

Engineering the chloroplast targeted malarial vaccine antigens in Chlamydomonas starch granules

Background

Malaria, an Anopheles-borne parasitic disease, remains a major global health problem causing illness and death that disproportionately affects developing countries. Despite the incidence of malaria, which remains one of the most severe infections of human populations, there is no licensed vaccine against this life-threatening disease. In this context, we decided to explore the expression of Plasmodium vaccine antigens fused to the granule bound starch synthase (GBSS), the major protein associated to the starch matrix in all starch-accumulating plants and algae such as Chlamydomonas reinhardtii.

Methods and Findings

We describe the development of genetically engineered starch granules containing plasmodial vaccine candidate antigens produced in the unicellular green algae Chlamydomonas reinhardtii. We show that the C-terminal domains of proteins from the rodent Plasmodium species, Plasmodium berghei Apical Major Antigen AMA1, or Major Surface Protein MSP1 fused to the algal granule bound starch synthase (GBSS) are efficiently expressed and bound to the polysaccharide matrix. Mice were either immunized intraperitoneally with the engineered starch particles and Freund adjuvant, or fed with the engineered particles co-delivered with the mucosal adjuvant, and challenged intraperitoneally with a lethal inoculum of P. Berghei. Both experimental strategies led to a significantly reduced parasitemia with an extension of life span including complete cure for intraperitoneal delivery as assessed by negative blood thin smears. In the case of the starch bound P. falciparum GBSS-MSP1 fusion protein, the immune sera or purified immunoglobulin G of mice immunized with the corresponding starch strongly inhibited in vitro the intra-erythrocytic asexual development of the most human deadly plasmodial species.

Conclusion

This novel system paves the way for the production of clinically relevant plasmodial antigens as algal starch-based particles designated herein as amylosomes, demonstrating that efficient production of edible vaccines can be genetically produced in Chlamydomonas.     

Paedomorphosis as an Evolutionary Driving Force: Insights from Deep-Sea Brittle Stars

Heterochronic development has been proposed to have played an important role in the evolution of echinoderms. In the class Ophiuroidea, paedomorphosis (retention of juvenile characters into adulthood) has been documented in the families Ophiuridae and Ophiolepididae but not been investigated on a broader taxonomic scale. Historical errors, confusing juvenile stages with paedomorphic species, show the difficulties in correctly identifying the effects of heterochrony on development and evolution. This study presents a detailed analysis of 40 species with morphologies showing various degrees of juvenile appearance in late ontogeny. They are compared to a range of early ontogenetic stages from paedomorphic and non-paedomorphic species. Both quantitative and qualitative measurements are taken and analysed. The results suggest that strongly paedomorphic species are usually larger than other species at comparable developmental stage. The findings support recent notions of polyphyletic origin of the families Ophiuridae and Ophiolepididae. The importance of paedomorphosis and its correct recognition for the practice of taxonomy and phylogeny are emphasized.     

Kaposi's Sarcoma Associated Herpesvirus Tegument Protein ORF75 Is Essential for Viral Lytic Replication and Plays a Critical Role in the Antagonization of ND10-Instituted Intrinsic Immunity

Nuclear domain 10 (ND10) components are restriction factors that inhibit herpesviral replication. Effector proteins of different herpesviruses can antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. We investigated the interplay of Kaposi''s Sarcoma-Associated Herpesvirus (KSHV) infection and cellular defense by nuclear domain 10 (ND10) components. Knock-down experiments in primary human cells show that KSHV-infection is restricted by the ND10 components PML and Sp100, but not by ATRX. After KSHV infection, ATRX is efficiently depleted and Daxx is dispersed from ND10, indicating that these two ND10 components can be antagonized by KSHV. We then identified the ORF75 tegument protein of KSHV as the viral factor that induces the disappearance of ATRX and relocalization of Daxx. ORF75 belongs to a viral protein family (viral FGARATs) that has homologous proteins in all gamma-herpesviruses. Isolated expression of ORF75 in primary cells induces a relocalization of PML and dispersal of Sp100, indicating that this viral effector protein is able to influence multiple ND10 components. Moreover, by constructing a KSHV mutant harboring a stop codon at the beginning of ORF75, we could demonstrate that ORF75 is absolutely essential for viral replication and the initiation of viral immediate-early gene expression. Using recombinant viruses either carrying Flag- or YFP-tagged variants of ORF75, we could further corroborate the role of ORF75 in the antagonization of ND10-mediated intrinsic immunity, and show that it is independent of the PML antagonist vIRF3. Members of the viral FGARAT family target different ND10 components, suggesting that the ND10 targets of viral FGARAT proteins have diversified during evolution. We assume that overcoming ND10 intrinsic defense constitutes a critical event in the replication of all herpesviruses; on the other hand, restriction of herpesviral replication by ND10 components may also promote latency as the default outcome of infection.     

A Recombination Hotspot in a Schizophrenia-Associated Region of GABRB2

Background

Schizophrenia is a major disorder with complex genetic mechanisms. Earlier, population genetic studies revealed the occurrence of strong positive selection in the GABRB2 gene encoding the β₂ subunit of GABA_A receptors, within a segment of 3,551 bp harboring twenty-nine single nucleotide polymorphisms (SNPs) and containing schizophrenia-associated SNPs and haplotypes.

Methodology/Principal Findings

In the present study, the possible occurrence of recombination in this ‘S1–S29’ segment was assessed. The occurrence of hotspot recombination was indicated by high resolution recombination rate estimation, haplotype diversity, abundance of rare haplotypes, recurrent mutations and torsos in haplotype networks, and experimental haplotyping of somatic and sperm DNA. The sub-segment distribution of relative recombination strength, measured by the ratio of haplotype diversity (H_d) over mutation rate (θ), was indicative of a human specific Alu-Yi6 insertion serving as a central recombining sequence facilitating homologous recombination. Local anomalous DNA conformation attributable to the Alu-Yi6 element, as suggested by enhanced DNase I sensitivity and obstruction to DNA sequencing, could be a contributing factor of the increased sequence diversity. Linkage disequilibrium (LD) analysis yielded prominent low LD points that supported ongoing recombination. LD contrast revealed significant dissimilarity between control and schizophrenic cohorts. Among the large array of inferred haplotypes, H26 and H73 were identified to be protective, and H19 and H81 risk-conferring, toward the development of schizophrenia.

Conclusions/Significance

The co-occurrence of hotspot recombination and positive selection in the S1–S29 segment of GABRB2 has provided a plausible contribution to the molecular genetics mechanisms for schizophrenia. The present findings therefore suggest that genome regions characterized by the co-occurrence of positive selection and hotspot recombination, two interacting factors both affecting genetic diversity, merit close scrutiny with respect to the etiology of common complex disorders.