The intestinal microbiota undergoes diurnal compositional and functional oscillations that affect metabolic homeostasis, but the mechanisms by which the rhythmic microbiota influences host circadian ...activity remain elusive. Using integrated multi-omics and imaging approaches, we demonstrate that the gut microbiota features oscillating biogeographical localization and metabolome patterns that determine the rhythmic exposure of the intestinal epithelium to different bacterial species and their metabolites over the course of a day. This diurnal microbial behavior drives, in turn, the global programming of the host circadian transcriptional, epigenetic, and metabolite oscillations. Surprisingly, disruption of homeostatic microbiome rhythmicity not only abrogates normal chromatin and transcriptional oscillations of the host, but also incites genome-wide de novo oscillations in both intestine and liver, thereby impacting diurnal fluctuations of host physiology and disease susceptibility. As such, the rhythmic biogeography and metabolome of the intestinal microbiota regulates the temporal organization and functional outcome of host transcriptional and epigenetic programs.
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•Intestinal microbiota biogeography and metabolome undergo diurnal oscillations•Circadian oscillations of serum metabolites are regulated by the microbiota•Microbiota rhythms program the circadian epigenetic and transcriptional landscape•The microbiota regulates the circadian liver transcriptome and detoxification pattern
Diurnal oscillations in microbial localization and metabolite production in the gut have a major impact on the circadian epigenetic and transcriptional landscape of host tissues, not only locally, but also at distant sites such as the liver.
Disulfide bond formation in secretory proteins occurs primarily in the endoplasmic reticulum (ER), where multiple enzyme families catalyze cysteine cross-linking. Quiescin sulfhydryl oxidase 1 ...(QSOX1) is an atypical disulfide catalyst, localized to the Golgi apparatus or secreted from cells. We examined the physiological function for extracellular catalysis of de novo disulfide bond formation by QSOX1. QSOX1 activity was required for incorporation of laminin into the extracellular matrix (ECM) synthesized by fibroblasts, and ECM produced without QSOX1 was defective in supporting cell-matrix adhesion. We developed an inhibitory monoclonal antibody against QSOX1 that could modulate ECM properties and undermine cell migration.
d-Serine is a coagonist of N-methyl-d-aspartate (NMDA) receptors that occurs at high levels in the brain. Biosynthesis of d-serine is carried out by serine racemase, which converts l-to d-serine. ...d-Serine has been demonstrated to occur in glial cells, leading to the proposal that astrocytes are the only source of d-serine. We now report significant amounts of serine racemase and d-serine in primary neuronal cultures and neurons in vivo. Several neuronal culture types expressed serine racemase, and d-serine synthesis was comparable with that in glial cultures. Immunohistochemical staining of brain sections with new antibodies revealed the presence of serine racemase and d-serine in neurons. Cortical neurons expressing serine racemase also expressed the NR2a subunit in situ. Neuron-derived d-serine contributes to NMDA receptor activation in cortical neuronal cultures. Degradation of endogenous d-serine by addition of the recombinant enzyme d-serine deaminase diminished NMDA-elicited excitotoxicity. Release of neuronal d-serine was mediated by ionotropic glutamate receptor agonists such as NMDA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate. Removal of either external Ca2+ or Na+ blocked d-serine release. Release of d-serine was mostly through a cytosolic route because it was insensitive to bafilomycin A1, a potent inhibitor of vesicular neurotransmitter uptake. d-Serine was also not transported into purified synaptic vesicles under conditions optimal for the uptake of known transmitters. Our results suggest that neurons are a major source of d-serine. Glutamate-induced neuronal d-serine release provides a novel mechanism for activating NMDA receptors by an autocrine or paracrine way.
T cell surfaces are covered with microvilli, actin-rich and flexible protrusions. We use super-resolution microscopy to show that ≥90% of T cell receptor (TCR) complex molecules TCRαβ and TCRζ, as ...well as the co-receptor CD4 (cluster of differentiation 4) and the co-stimulatory molecule CD2, reside on microvilli of resting human T cells. Furthermore, TCR proximal signaling molecules involved in the initial stages of the immune response, including the protein tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase) and the key adaptor LAT (linker for activation of T cells), are also enriched on microvilli. Notably, phosphorylated proteins of the ERM (ezrin, radixin, and moesin) family colocalize with TCRαβ as well as with actin filaments, implying a role for one or more ERMs in linking the TCR complex to the actin cytoskeleton within microvilli. Our results establish microvilli as key signaling hubs, in which the TCR complex and its proximal signaling molecules and adaptors are preassembled prior to activation in an ERM-dependent manner, facilitating initial antigen sensing.
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•Single-molecule microscopy maps signaling molecules on the resting T cell membrane•TCRαβ, TCRζ, CD4, CD2, Lck, and LAT are all highly enriched on microvilli•TCR localization is mediated by ERM proteins•Microvilli are hubs for TCR signaling, facilitating fast initial immune response
T-cell surfaces are covered with microvilli, actin-rich and flexible protrusions. Ghosh et al. show that T cell receptor complex molecules, as well as several proximal signaling molecules, are preassembled on microvilli in an ERM-dependent manner. These results establish microvilli as key signaling hubs that facilitate initial antigen sensing by T cells.
D-serine occurs at high levels in the brain, where it is an endogenous coagonist at the "glycine site" of NMDA receptors. However, D-serine action has not been previously compared with that of ...endogenous glycine, and the relative importance of the two coagonists remains unclear. We now investigated the efficiencies of the two coagonists in mediating NMDA receptor neurotoxicity in organotypic hippocampal slices. Removal of endogenous D-serine from slices was achieved by pretreating the tissue with recombinant D-serine deaminase enzyme. This enzyme is several orders of magnitude more efficient than previous methods to remove D-serine. We report that complete removal of D-serine virtually abolished NMDA-elicited neurotoxicity but did not protect against kainate. Although levels of glycine were 10-fold higher than D-serine, endogenous glycine was ineffective in mediating NMDA receptor neurotoxicity. The effect of endogenous glycine could be observed only after simultaneous removal of endogenous D-serine and blockage of the glycine transporter GlyT1. Our data indicate that D-serine is the dominant coagonist for NMDA receptor-elicited neurotoxicity, mediating all cell death elicited by NMDA in organotypic slices. The results suggest an essential role for this unusual D-amino acid, with implications for the mechanism of neuronal death in the nervous system.
The recent discovery of multiple giant double-stranded DNA (dsDNA) viruses blurred the consensual distinction between viruses and cells due to their size, as well as to their structural and genetic ...complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, termed "viral factories," where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive postinfection time points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics, and imaging analyses. These analyses revealed that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as "production lines" of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses.
Large dsDNA viruses such as vaccinia virus and the giant mimivirus, as well as many positive-strand RNA viruses, generate elaborate cytoplasmic organelles in which the multiple and diverse transactions required for viral replication and assembly occur. These organelles, which were termed "viral factories," are attracting much interest due to the increasing realization that the rapid and continuous production of viral progeny is a direct outcome of the elaborate structure and composition of the factories, which act as efficient production lines. To get new insights into the nature and function of viral factories, we devised a method that allows, for the first time, the isolation of these organelles. Analyses of the isolated factories generated at different times postinfection by mass spectrometry-based proteomics provide new perceptions of their role and reveal the highly dynamic nature of these organelles.
This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, ...methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.
Historically, multicellular bacterial communities, known as biofilms, have been thought to be held together solely by a self-produced extracellular matrix. Our study identified a novel mechanism ...maintaining
and
biofilms-active production of calcite minerals. We studied, for the first time, the effects of mutants defective in biomineralization and calcite formation on biofilm development, resilience and morphology. We demonstrated that an intrinsic rise in carbon dioxide levels within the biofilm is a strong trigger for the initiation of calcite-dependent patterning. The calcite-dependent patterns provide resistance to environmental insults and increase the overall fitness of the microbial community. Our results suggest that it is highly feasible that the formation of mineral scaffolds plays a cardinal and conserved role in bacterial multicellularity.
D-Serine is thought to be a glia-derived transmitter that activates N-methyl D-aspartate receptors (NMDARs) in the brain. Here, we investigate the pathways for D-serine release using primary ...cultures, brain slices, and in vivo microdialysis. In contrast with the notion that D-serine is exclusively released from astrocytes, we found that D-serine is released by neuronal depolarization both in vitro and in vivo. Veratridine (50 μM) or depolarization by 40 mM KCl elicits a significant release of endogenous D-serine from primary neuronal cultures. Controls with astrocyte cultures indicate that glial cells are insensitive to veratridine, but release D-serine mainly by the opening of volume-regulated anion channels. In cortical slices perfused with veratridine, endogenous D-serine release is 10-fold higher than glutamate receptor-evoked release. Release of D-serine from slices does not require internal or external Ca²⁺, suggesting a nonvesicular release mechanism. To confirm the neuronal origin of D-serine, we selectively loaded neurons in cortical slices with D-³Hserine or applied D-alanine, which specifically releases D-serine from neurons. Depolarization with veratridine promotes D-serine release in vivo monitored by high temporal resolution microdialysis of the striatum. Our data indicate that the neuronal pool of D-serine plays a major role in D-serine dynamics, with implications for the regulation of NMDAR transmission. Rosenberg, D., Kartvelishvily, E., Shleper, M., Klinker, C. M. C., Bowser, M. T., Wolosker, H. Neuronal release of D-serine: a physiological pathway controlling extracellular D-serine concentration.
is a bloom-forming microalga that affects the global sulfur cycle by producing large amounts of dimethylsulfoniopropionate (DMSP) and its volatile metabolic product dimethyl sulfide. Top-down ...regulation of
blooms has been attributed to viruses and grazers; however, the possible involvement of algicidal bacteria in bloom demise has remained elusive. We demonstrate that a
strain,
D7, that we isolated from a North Atlantic
bloom, exhibited algicidal effects against
upon coculturing. Both the alga and the bacterium were found to co-occur during a natural
bloom, therefore establishing this host-pathogen system as an attractive, ecologically relevant model for studying algal-bacterial interactions in the oceans. During interaction,
D7 consumed and metabolized algal DMSP to produce high amounts of methanethiol, an alternative product of DMSP catabolism. We revealed a unique strain-specific response, in which
strains that exuded higher amounts of DMSP were more susceptible to
D7 infection. Intriguingly, exogenous application of DMSP enhanced bacterial virulence and induced susceptibility in an algal strain typically resistant to the bacterial pathogen. This enhanced virulence was highly specific to DMSP compared to addition of propionate and glycerol which had no effect on bacterial virulence. We propose a novel function for DMSP, in addition to its central role in mutualistic interactions among marine organisms, as a mediator of bacterial virulence that may regulate
blooms.
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