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The metabolism of seaweeds depends on environmental parameters, the availability of nutrients, and biotic/abiotic stresses, therefore, their chemical composition fluctuates throughout the year. This study investigated seasonal variations in the metabolome of the Baltic Sea brown alga Fucus vesiculosus and its potential relation to the bioactivity profile. By using a definitive screening design (DSD) combined with pressurised liquid extraction (PLE), an optimised protocol was developed to extract algal biomass monthly for a full calendar year. An untargeted metabolomics approach using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MSn)-based molecular networking and manual dereplication was employed. The extracts were simultaneously screened for their in vitro antimicrobial, anticancer/apoptotic, and free radical scavenging activities. 44 compounds were putatively dereplicated in the metabolome. Many compounds were found to vary with the sampling month, phlorotannin total ion count (TIC) was highest in summer, whilst chlorophylls, lipids, and carotenoids peaked in winter and spring. The greatest radical scavenging and apoptotic activities against pancreas cancer cells observed in the summer months were attributed to high phlorotannin TIC. Methicillin-resistant Staphylococcus aureus (MRSA) inhibitory activity was produced year-round without a clear seasonal trend. This is the first study applying DSD-based optimised PLE extraction combined with a metabolome analysis of F. vesiculosus for the identification of seasonal variations in both metabolome and bioactivity.

International audience; The metabolism of seaweeds depends on environmental parameters, the availability of nutrients, and biotic/abiotic stresses; therefore, their chemical composition fluctuates throughout the year. This study investigated seasonal variations in the metabolome of the Baltic Sea brown alga Fucus vesiculosus and its potential relation to the bioactivity profile. By using a definitive screening design (DSD) combined with pressurised liquid extraction (PLE), an optimised protocol was developed to extract algal biomass monthly for a full calendar year. An untargeted metabolomics approach using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MSn)-based molecular networking and manual dereplication was employed. The extracts were simultaneously screened for their in vitro antimicrobial, anticancer/apoptotic, and free radical scavenging activities. 44 compounds were putatively dereplicated in the metabolome. Many compounds were found to vary with the sampling month; phlorotannin total ion count (TIC) was highest in summer, whilst chlorophylls, lipids, and carotenoids peaked in winter and spring. The greatest radical scavenging and apoptotic activities against pancreas cancer cells observed in the summer months were attributed to high phlorotannin TIC. Methicillin-resistant Staphylococcus aureus (MRSA) inhibitory activity was produced year-round without a clear seasonal trend. This is the first study applying DSD-based optimised PLE extraction combined with a metabolome analysis of F. vesiculosus for the identification of seasonal variations in both metabolome and bioactivity.

Human cytomegalovirus (HCMV) encodes four G protein-coupled receptor (GPCR) homologs, termed pUS27, pUS28, pUL33, and pUL78. In contrast to the extensively characterized vGPCRs pUS28 and pUL33, knowledge concerning pUS27 and pUL78 is limited. Previous studies already demonstrated constitutive internalization of pUS27 and pUL78, as well as an association with the endosomal machinery, however, these results were mainly obtained using transiently transfected cells. To explore the subcellular localization of both receptors during viral infection, we constructed recombinant HCMVs expressing tagged vGPCRs. Colocalization analyses revealed a predominant association of pUS27 or pUL78 with the trans-Golgi network or the endoplasmic reticulum, respectively. Intriguingly, our data emphasize that protein sorting is highly regulated by viral functions as we detected dramatic changes in the colocalization of pUS27 and pUL78 with endosomal markers during progression of HCMV replication. Furthermore, we observed cell type-dependent differences in trafficking of both vGPCRs between fibroblasts and epithelial cells. Most importantly, infection experiments with a recombinant HCMV carrying tagged versions of pUS27 and pUL78 simultaneously, revealed that these two proteins do not colocalize during viral infection. This contrasts to results of transient expression experiments. In conclusion, our results highlight the importance to investigate vGPCR trafficking in a viral context.

BackgroundSome herpesviruses like human cytomegalovirus (HCMV) encode viral G protein-coupled receptors that cause reprogramming of cell signaling to facilitate dissemination of the virus, prevent immune surveillance and establish life-long latency. Human GPCRs are known to function in complex signaling networks involving direct physical interactions as well as indirect crosstalk of orthogonal signaling networks. The human chemokine receptor CXCR4 is expressed on hematopoietic stem cells, leukocytes, endothelial and epithelial cells, which are infected by HCMV or display reservoirs of latency.ResultsWe investigated the potential heteromerization of US28 with CXCR4 as well as the influence of US28 on CXCR4 signaling. Using Bioluminescence Resonance Energy Transfer and luciferase-complementation based methods we show that US28 expression exhibits negative effects on CXCR4 signaling and constitutive surface expression in HEK293T cells. Furthermore, we demonstrate that this effect is not mediated by receptor heteromerization but via signaling crosstalk. Additionally, we show that in HCMV, strain TB40E, infected HUVEC the surface expression of CXCR4 is strongly downregulated, whereas in TB40E-delUS28 infected cells, CXCR4 surface expression is not altered in particular at late time points of infection.ConclusionsWe show that the vGPCR US28 is leading to severely disturbed signaling and surface expression of the chemokine receptor CXCR4 thereby representing an effective mechanism used by vGPCRs to reprogram host cell signaling. In contrast to other studies, we demonstrate that these effects are not mediated via heteromerization.