Protein synthesis persists during necrotic cell death

Cell death is an intrinsic part of metazoan development and mammalian immune regulation. Whereas the molecular events orchestrating apoptosis have been characterized extensively, little is known about the biochemistry of necrotic cell death. Here, we show that, in contrast to apoptosis, the induction of necrosis does not lead to the shut down of protein synthesis. The rapid drop in protein synthesis observed in apoptosis correlates with caspase-dependent breakdown of eukaryotic translation initiation factor (eIF) 4G, activation of the double-stranded RNA-activated protein kinase PKR, and phosphorylation of its substrate eIF2-α. In necrosis induced by tumor necrosis factor, double-stranded RNA, or viral infection, de novo protein synthesis persists and 28S ribosomal RNA fragmentation, eIF2-α phosphorylation, and proteolytic activation of PKR are absent. Collectively, these results show that, in contrast to apoptotic cells, necrotic dying cells retain the opportunity to synthesize proteins

The effect of hypoxia on Daphnia magna performance and its associated microbial and bacterioplankton community: A scope for phenotypic plasticity and microbiome community interactions upon environmental stress?

The depletion of oxygen as a result of increased stratification and decreased oxygen solubility is one of the most significant chemical changes occurring in aquatic ecosystems as a result of global environmental change. Hence, more aquatic organisms will be exposed to hypoxic conditions over time. Deciphering the effects of hypoxia on strong ecological interactors in this ecosystem’s food web is critical for predicting how aquatic communities can respond to such an environmental disturbance. Here (sub-)lethal effects of hypoxia and whether these are genotype specific in Daphnia, a keystone species of freshwater ecosystems, are studied. This is especially relevant upon studying genetic responses with respect to phenotypic switches upon environmental stress. Further, we investigated the effect of hypoxia on the Daphnia microbial community to test if the microbiome plays a role in the phenotypic switch and tolerance to hypoxia. For this, two Daphnia genotypes were exposed for two weeks to either hypoxia or normoxia and host performance was monitored together with changes in the host associated and free-living microbial community after this period. We detected phenotypic plasticity for some of the tested Daphnia performance traits. The microbial community of the bacterioplankton and Daphnia associated microbial community responded via changes in species richness and community composition and structure. The latter response was different for the two genotypes suggesting that the microbiome plays an important role in phenotypic plasticity with respect to hypoxia tolerance in Daphnia, but further testing (e.g., through microbiome transplants) is needed to confirm this

Synergistic effects of dual parasitism in Daphnia magna under nutrient limitation

Human-induced increases in the bioavailability of carbon (C), nitrogen (N) and phosphorus (P) has the potential to alter the context for host-parasite dynamics in aquatic ecosystems. Given that both eutrophication and infectious diseases are becoming more prominent, it is essential to disentangle the factors that determine virulence expression in keystone grazers. Here, we focus on the impact of nutrient limitation in single versus multiple parasite exposure in the water flea Daphnia magna (Crustacea, Branchiopoda). For this, we fed Daphnia with algae differing in C:N:P ratio and exposed them to two virulent parasites, Pasteuria ramosa (Bacteria) and White Fat Cell (WFCD, unknown classification), both in single and multiple parasite exposure treatments. Multiple parasite exposure synergistically interacted to reduce host survival, especially under severe nutrient limitation, which was mainly driven by WFCD exposure. At the same time, Daphnia shifted the time to start reproducing forward, which resulted in a higher reproductive output upon multiple parasite exposure. We here discuss these results within the framework of host stress responses, nutrient allocation and energy budgets and conclude that how food quality interferes with host-parasite interactions depends on the parasite species studied, the nutrient requirements of all actors and the trait investigated.status: publishe

Microbiome mediated tolerance to biotic stressors: a case study of the interaction between a toxic cyanobacterium and an oomycete-like infection in Daphnia magna

Organisms are increasingly facing multiple, potentially interacting stressors in natural populations. The ability of populations coping with combined stressors depends on their tolerance to individual stressors and how stressors interact, which may not be correctly captured in controlled laboratory settings. One largely unexplored reason for this is that the microbial communities in laboratory settings often differ from the natural environment, which could result in different stressor responses and interaction patterns. In this study, we investigated the impact of single and combined exposure to a toxic cyanobacterium and an oomycete-like parasite on the performance of three Daphnia magna genotypes. Daphnia individuals were first sterilized and then experimentally given a natural or a laboratory-derived microbial inoculum. Survival, reproduction and body size were monitored for three weeks and gut microbiomes were sampled and characterized at the end of the experiment. Our study confirmed that natural and laboratory microbial inocula and gut microbiomes are differently structured with natural microbiomes being more diverse than laboratory microbiomes. Our results showed that exposure to the stressors reduced D. magna performance compared to the control. An antagonistic interaction between the two biotic stressors was revealed with respect to D. magna survival, when Daphnia individuals were exposed to the laboratory microbial inoculum. This effect was consistent across all three genotypes. In Daphnia exposed to a natural microbial inoculum this antagonistic interaction could not be detected and the genotype x exposure interaction was genotype dependent. Our results indicate that host-stressor interactions depend on the microbial inoculum and that the gut microbiome has potentially a strong role in this, thereby providing an unexplored dimension to multiple-stressor research

Diet and genotype of an aquatic invertebrate affect the composition of free-living microbial communities

In spite of the growing interest in the role of the gut microbiome (GM) in host physiology and health, the mechanisms governing its assembly and its effects on the environment are poorly understood. In this article, we show that the host genotype and the GM of Daphnia influence the community structure of the surrounding bacterioplankton (BPK). When Daphnia genotypes were placed in an identical environment, both the GM and BPK showed a genotype and diet-dependent taxonomic composition. Overall, the GM strongly differed from the BPK in taxonomic composition and was characterized by a lower alpha-diversity, suggesting a selective rejecting of bacteria from the regional species pool. In a microbiome transplant experiment, the assembly of both the GM and BPK was strongly affected by the host genotype and the inoculum to which germ-free Daphnia were exposed. The combination of these results suggests a strong interaction between the host genotype, its GM and free-living microbial communities. Currently, it is generally assumed that an animal's diet has a strong effect on the animal's GM, but only a negligible (if any) effect on the surrounding environment. However, our results indicate that the diet/microbiome inocula have a small effect on the gut community and a large effect on the community in the surrounding environment. This structuring genotype x microbiome x environment effect is an essential prerequisite that could indicate that microbiomes play an important role in eco-evolutionary processes

Diet and Genotype of an Aquatic Invertebrate Affect the Composition of Free-Living Microbial Communities

International audienceIn spite of the growing interest in the role of the gut microbiome (GM) in host physiology and health, the mechanisms governing its assembly and its effects on the environment are poorly understood. In this article, we show that the host genotype and the GM of Daphnia influence the community structure of the surrounding bacterioplankton (BPK). When Daphnia genotypes were placed in an identical environment, both the GM and BPK showed a genotype and diet-dependent taxonomic composition. Overall, the GM strongly differed from the BPK in taxonomic composition and was characterized by a lower α-diversity, suggesting a selective rejecting of bacteria from the regional species pool. In a microbiome transplant experiment, the assembly of both the GM and BPK was strongly affected by the host genotype and the inoculum to which germ-free Daphnia were exposed. The combination of these results suggests a strong interaction between the host genotype, its GM and free-living microbial communities. Currently, it is generally assumed that an animal's diet has a strong effect on the animal's GM, but only a negligible (if any) effect on the surrounding environment. However, our results indicate that the diet/microbiome inocula have a small effect on the gut community and a large effect on the community in the surrounding environment. This structuring genotype × microbiome × environment effect is an essential prerequisite that could indicate that microbiomes play an important role in eco-evolutionary processes

Daphnia magna tolerance to toxic cyanobacteria in the presence of an opportunistic infection within an evolutionary perspective

status: publishe

Data_Sheet_1_The effect of hypoxia on Daphnia magna performance and its associated microbial and bacterioplankton community: A scope for phenotypic plasticity and microbiome community interactions upon environmental stress?.pdf

The depletion of oxygen as a result of increased stratification and decreased oxygen solubility is one of the most significant chemical changes occurring in aquatic ecosystems as a result of global environmental change. Hence, more aquatic organisms will be exposed to hypoxic conditions over time. Deciphering the effects of hypoxia on strong ecological interactors in this ecosystem’s food web is critical for predicting how aquatic communities can respond to such an environmental disturbance. Here (sub-)lethal effects of hypoxia and whether these are genotype specific in Daphnia, a keystone species of freshwater ecosystems, are studied. This is especially relevant upon studying genetic responses with respect to phenotypic switches upon environmental stress. Further, we investigated the effect of hypoxia on the Daphnia microbial community to test if the microbiome plays a role in the phenotypic switch and tolerance to hypoxia. For this, two Daphnia genotypes were exposed for two weeks to either hypoxia or normoxia and host performance was monitored together with changes in the host associated and free-living microbial community after this period. We detected phenotypic plasticity for some of the tested Daphnia performance traits. The microbial community of the bacterioplankton and Daphnia associated microbial community responded via changes in species richness and community composition and structure. The latter response was different for the two genotypes suggesting that the microbiome plays an important role in phenotypic plasticity with respect to hypoxia tolerance in Daphnia, but further testing (e.g., through microbiome transplants) is needed to confirm this.</p

Targeted Peptidecentric Proteomics Reveals Caspase-7 as a Substrate of the Caspase-1 Inflammasomes *S⃞

The aspartate-specific cysteine protease caspase-1 is activated by the inflammasomes and is responsible for the proteolytic maturation of the cytokines IL-1β and IL-18 during infection and inflammation. To discover new caspase-1 substrates, we made use of a proteome-wide gel-free differential peptide sorting methodology that allows unambiguous localization of the processing site in addition to identification of the substrate. Of the 1022 proteins that were identified, 20 were found to be specifically cleaved after Asp in the setup incubated with recombinant caspase-1. Interestingly, caspase-7 emerged as one of the identified caspase-1 substrates. Moreover half of the other identified cleavage events occurred at sites closely resembling the consensus caspase-7 recognition sequence DEVD, suggesting caspase-1-mediated activation of endogenous caspase-7 in this setup. Consistently recombinant caspase-1 cleaved caspase-7 at the canonical activation sites Asp23 and Asp198, and recombinant caspase-7 processed a subset of the identified substrates. In vivo, caspase-7 activation was observed in conditions known to induce activation of caspase-1, including Salmonella infection and microbial stimuli combined with ATP. Interestingly Salmonella- and lipopolysaccharide + ATP-induced activation of caspase-7 was abolished in macrophages deficient in caspase-1, the pattern recognition receptors Ipaf and Cryopyrin, and the inflammasome adaptor ASC, demonstrating an upstream role for the caspase-1 inflammasomes in caspase-7 activation in vivo. In contrast, caspase-1 and the inflammasomes were not required for caspase-3 activation. In conclusion, we identified 20 new substrates activated downstream of caspase-1 and validated caspase-1-mediated caspase-7 activation in vitro and in knock-out macrophages. These results demonstrate for the first time the existence of a nucleotide binding and oligomerization domain-like receptor/caspase-1/caspase-7 cascade and the existence of distinct activation mechanisms for caspase-3 and -7 in response to microbial stimuli and bacterial infection