Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication

Cytotoxic necrotizing factors (CNFs) are bacterial single‐chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three‐dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full‐length Yersinia pseudotuberculosis CNF_Y. CNF_Y consists of five domains (D1–D5), and by integrating structural and functional data, we demonstrate that D1–3 act as export and translocation module for the catalytic unit (D4–5) and for a fused β‐lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP‐ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4–5 fragment. This liberates D5 from a semi‐blocked conformation in full‐length CNF_Y, leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad‐specificity protein delivery tool.     

Dissociated presenilin-1 and TACE processing of ErbB4 in lung alveolar type II cell differentiation

Neuregulin (NRG) stimulation of ErbB4 signaling is important for type II cell surfactant synthesis. ErbB4 may mediate gene expression via a non-canonical pathway involving enzymatic cleavage releasing its intracellular domain (4ICD) for nuclear trafficking and gene regulation. The accepted model for release of 4ICD is consecutive cleavage by Tumor necrosis factor alpha Converting Enzyme (TACE) and γ-secretase enzymes. Here, we show that 4ICD mediates surfactant synthesis and its release by γ-secretase is not dependent on previous TACE cleavage. We used siRNA to silence Presenilin-1 (PSEN-1) expression in a mouse lung type II epithelial cell line (MLE12 cells), and both siRNA knockdown and chemical inhibition of TACE. Knockdown of PSEN-1 significantly decreased baseline and NRG-stimulated surfactant phospholipid synthesis, expression of the surfactant proteins SP-B and SP-C, as well as 4ICD levels, with no change in ErbB4 ectodomain shedding. Neither siRNA knockdown nor chemical inhibition of TACE inhibited 4ICD release or surfactant synthesis. PSEN-1 cleavage of ErbB4 for non-canonical signaling through 4ICD release does not require prior cleavage by TACE.     

Specific Gene Expression Responses to Parasite Genotypes Reveal Redundancy of Innate Immunity in Vertebrates

Vertebrate innate immunity is the first line of defense against an invading pathogen and has long been assumed to be largely unspecific with respect to parasite/pathogen species. However, recent phenotypic evidence suggests that immunogenetic variation, i.e. allelic variability in genes associated with the immune system, results in host-parasite genotype-by-genotype interactions and thus specific innate immune responses. Immunogenetic variation is common in all vertebrate taxa and this reflects an effective immunological function in complex environments. However, the underlying variability in host gene expression patterns as response of innate immunity to within-species genetic diversity of macroparasites in vertebrates is unknown. We hypothesized that intra-specific variation among parasite genotypes must be reflected in host gene expression patterns. Here we used high-throughput RNA-sequencing to examine the effect of parasite genotypes on gene expression patterns of a vertebrate host, the three-spined stickleback (Gasterosteus aculeatus). By infecting naïve fish with distinct trematode genotypes of the species Diplostomum pseudospathaceum we show that gene activity of innate immunity in three-spined sticklebacks depended on the identity of an infecting macroparasite genotype. In addition to a suite of genes indicative for a general response against the trematode we also find parasite-strain specific gene expression, in particular in the complement system genes, despite similar infection rates of single clone treatments. The observed discrepancy between infection rates and gene expression indicates the presence of alternative pathways which execute similar functions. This suggests that the innate immune system can induce redundant responses specific to parasite genotypes.     

Glutathione contributes to plant defence against parasitic cyst nematodes

Cyst nematodes (CNs) are an important group of root-infecting sedentary endoparasites that severely damage many crop plants worldwide. An infective CN juvenile enters the host's roots and migrates towards the vascular cylinder, where it induces the formation of syncytial feeding cells, which nourish the CN throughout its parasitic stages. Here, we examined the role of glutathione (l -γ-glutamyl-l -cysteinyl-glycine) in Arabidopsis thaliana on infection with the CN Heterodera schachtii. Arabidopsis lines with mutations pad2, cad2, or zir1 in the glutamate–cysteine ligase (GSH1) gene, which encodes the first enzyme in the glutathione biosynthetic pathway, displayed enhanced CN susceptibility, but susceptibility was reduced for rax1, another GSH1 allele. Biochemical analysis revealed differentially altered thiol levels in these mutants that was independent of nematode infection. All glutathione-deficient mutants exhibited impaired activation of defence marker genes as well as genes for biosynthesis of the antimicrobial compound camalexin early in infection. Further analysis revealed a link between glutathione-mediated plant resistance to CN infection and the production of camalexin on nematode infection. These results suggest that glutathione levels affect plant resistance to CN by fine-tuning the balance between the cellular redox environment and the production of compounds related to defence against infection.     

Vaccination Management and Vaccination Errors: A Representative Online-Survey among Primary Care Physicians

Background

Effective immunizations require a thorough, multi-step process, yet few studies comprehensively addressed issues around vaccination management.

Objectives

To assess variations in vaccination management and vaccination errors in primary care.

Methods

A cross sectional, web-based questionnaire survey was performed among 1157 primary physicians from North Rhine-Westphalia, Germany: a representative 10% random sample of general practitioners (n = 946) and all teaching physicians from the University Duisburg-Essen (n = 211). Four quality aspects with three items each were included: patient-related quality (patient information, patient consent, strategies to increase immunization rates), vaccine-related quality (practice vaccine spectrum, vaccine pre-selection, vaccination documentation), personnel-related quality (recommendation of vaccinations, vaccine application, personnel qualification) and storage-related quality (storage device, temperature log, vaccine storage control). For each of the four quality aspects, “good quality” was reached if all three criteria per quality aspect were fulfilled. Good vaccination management was defined as fulfilling all twelve items. Additionally, physicians’ experiences with errors and nearby-errors in vaccination management were obtained.

Results

More than 20% of the physicians participated in the survey. Good vaccination management was reached by 19% of the practices. Patient-related quality was good in 69% of the practices, vaccine-related quality in 73%, personnel-related quality in 59% and storage-related quality in 41% of the practices. No predictors for error reporting and good vaccination management were identified.

Conclusions

We identified good results for vaccine- and patient-related quality but need to improve issues that revolve around vaccine storage.     

Spying on your neighbours? Social information affects timing of breeding and stress hormone levels in a colonial seabird

A good overlap between offspring energetic requirements and availability of resources is required for successful reproduction. Accordingly, individuals from numerous species fine-tune their timing of breeding by integrating cues that predict environmental conditions during the offspring period. Besides acquiring information from their direct interaction with the environment (personal information), individuals can integrate information by observing the behaviours or performance of others (social information). The use of social information is often beneficial because the accumulated knowledge of conspecifics may represent a source of information more reliable than the intrinsically more limited personal information. However, although social information constitutes the major source of information in a wide range of contexts, studies investigating its use in the context of timing of breeding are scarce. We investigated whether black-legged kittiwakes (Rissa tridactyla) used social information to adjust the timing of egg-laying. We manipulated social information using a food-supplementation experiment, known to advance kittiwakes' reproductive phenology. We expected food-supplemented and unsupplemented pairs to delay and advance, respectively, their timing of laying when surrounded by a majority of neighbours from the opposite food-treatment. However, both unsupplemented and food-supplemented kittiwakes delayed egg-laying when surrounded by a higher proportion of neighbours from the opposite food-treatment. This result shows that kittiwakes use social information to time egg-laying, but that it is not used to match the seasonal peak of food availability. We suggest that when social and personal cues give contradictory environmental information, individuals may benefit from delaying laying to gather more information to make better decisions about investment into eggs. Further, we explored a potential proximate mechanism for the pattern we report. We show that baseline corticosterone, known to mediate reproductive decisions, was lower in unsupplemented females facing a higher proportion of food-supplemented neighbours. Altogether, our results suggest that to fine-tune their timing of laying, kittiwakes use complex decision-making processes in which social and personal information interplay.

     

Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes

Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable.     

Impact of Vitamin B12 on Formation of the Tetrachloroethene Reductive Dehalogenase in Desulfitobacterium hafniense Strain Y51

Corrinoids are essential cofactors of reductive dehalogenases in anaerobic bacteria. Microorganisms mediating reductive dechlorination as part of their energy metabolism are either capable of de novo corrinoid biosynthesis (e.g., Desulfitobacterium spp.) or dependent on exogenous vitamin B₁₂ (e.g., Dehalococcoides spp.). In this study, the impact of exogenous vitamin B₁₂ (cyanocobalamin) and of tetrachloroethene (PCE) on the synthesis and the subcellular localization of the reductive PCE dehalogenase was investigated in the Gram-positive Desulfitobacterium hafniense strain Y51, a bacterium able to synthesize corrinoids de novo. PCE-depleted cells grown for several subcultivation steps on fumarate as an alternative electron acceptor lost the tetrachloroethene-reductive dehalogenase (PceA) activity by the transposition of the pce gene cluster. In the absence of vitamin B₁₂, a gradual decrease of the PceA activity and protein amount was observed; after 5 subcultivation steps with 10% inoculum, more than 90% of the enzyme activity and of the PceA protein was lost. In the presence of vitamin B₁₂, a significant delay in the decrease of the PceA activity with an ∼90% loss after 20 subcultivation steps was observed. This corresponded to the decrease in the pceA gene level, indicating that exogenous vitamin B₁₂ hampered the transposition of the pce gene cluster. In the absence or presence of exogenous vitamin B₁₂, the intracellular corrinoid level decreased in fumarate-grown cells and the PceA precursor formed catalytically inactive, corrinoid-free multiprotein aggregates. The data indicate that exogenous vitamin B₁₂ is not incorporated into the PceA precursor, even though it affects the transposition of the pce gene cluster.     

A comment on the Hill–Turney exchange: from normative antagonism to interdisciplinary collaboration

Amniotic fluid-derived stem (AFS) cells were the first non-embryonic source of pluripotent stem cells discussed in Italy. AFS cells have been defined “ethical stem cells”, a socio-technical label denoting an array of cellular reprogramming techniques, biological artifacts, and somatic stem cells which make it possible to obtain pluripotent stem cells while avoiding the use of human embryos. The alleged pluripotent status of these cells triggered a debate in which pluripotency was re-positioned as the most promising feature of stem cells. This debate was characterized by discursive articulations in which the boundary between multipotency and pluripotency was blurred and a discourse on a duopoly of pluripotency (pertaining both to embryonic and non-embryonic stem cells) ensued. Drawing on qualitative discourse analysis of articles in scientific journals and newspapers, this paper explores the cultural meaning and political uses of these discursive articulations in the Italian stem cell research regulation debate. This paper also examines a set of aporias emerging from the attempt to incorporate ethical stances into the biological ontology of ethical stem cells, and the problem of constructing normativities in the biotechnology field.     

Proton Transfer to Flavin Stabilizes the Signaling State of the Blue Light Receptor Plant Cryptochrome

Plant cryptochromes regulate the circadian rhythm, flowering time, and photomorphogenesis in higher plants as responses to blue light. In the dark, these photoreceptors bind oxidized FAD in the photolyase homology region (PHR). Upon blue light absorption, FAD is converted to the neutral radical state, the likely signaling state, by electron transfer via a conserved tryptophan triad and proton transfer from a nearby aspartic acid. Here we demonstrate, by infrared and time-resolved UV-visible spectroscopy on the PHR domain, that replacement of the aspartic acid Asp-396 with cysteine prevents proton transfer. The lifetime of the radical is decreased by 6 orders of magnitude. This short lifetime does not permit to drive conformational changes in the C-terminal extension that have been associated with signal transduction. Only in the presence of ATP do both the wild type and mutant form a long-lived radical state. However, in the mutant, an anion radical is formed instead of the neutral radical, as found previously in animal type I cryptochromes. Infrared spectroscopic experiments demonstrate that the light-induced conformational changes of the PHR domain are conserved in the mutant despite the lack of proton transfer. These changes are not detected in the photoreduction of the non-photosensory d-amino acid oxidase to the anion radical. In conclusion, formation of the anion radical is sufficient to generate a protein response in plant cryptochromes. Moreover, the intrinsic proton transfer is required for stabilization of the signaling state in the absence of ATP.