Quantitative Proteomic Analysis of Outer Membrane Vesicles from Fusobacterium nucleatum Cultivated in the Mimic Cancer Environment.

Fusobacterium nucleatum is a Gram-negative bacterium that has been identified as an important pathogenic gut bacterium associated with colorectal cancer. Compared with the normal intestine, the pH value of the tumor microenvironment is weakly acidic. The metabolic changes of F. nucleatum in the tumor microenvironment, especially the protein composition of its outer membrane vesicles, remain unclear. Here, we systematically analyzed the effect of environmental pH on the proteome of outer membrane vesicles (OMVs) from F. nucleatum by tandem mass tag (TMT) labeling-high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A total of 991 proteins were identified in acidic OMVs (aOMVs) and neutral OMVs (nOMVs), including known virulence proteins and putative virulence proteins. Finally, 306 upregulated proteins and 360 downregulated proteins were detected in aOMVs, and approximately 70% of the expression of OMV proteins was altered under acidic conditions. A total of 29 autotransporters were identified in F. nucleatum OMVs, and 13 autotransporters were upregulated in aOMVs. Interestingly, three upregulated autotransporters (D5REI9, D5RD69, and D5RBW2) show homology to the known virulence factor Fap2, suggesting that they may be involved in various pathogenic pathways such as the pathway for binding with colorectal cancer cells. Moreover, we found that more than 70% of MORN2 domain-containing proteins may have toxic effects on host cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses demonstrated that a number of proteins were significantly enriched in multiple pathways involving fatty acid synthesis and butyrate synthesis. Seven metabolic enzymes involved in fatty acid metabolism pathways were identified in the proteomic data, of which 5 were upregulated and 2 were downregulated in aOMVs, while 14 metabolic enzymes involved in the butyric acid metabolic pathway were downregulated in aOMVs. In conclusion, we found a key difference in virulence proteins and pathways in the outer membrane vesicles of F. nucleatum between the tumor microenvironment pH and normal intestinal pH, which provides new clues for the prevention and treatment of colorectal cancer. IMPORTANCE F. nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. OMVs have been demonstrated to play key roles in pathogenesis by delivering toxins and other virulence factors to host cells. By employing quantitative proteomic analysis, we found that the pH conditions could affect the protein expression of the outer membrane vesicles of F. nucleatum. Under acidic conditions, approximately 70% of the expression of proteins in OMVs was altered. Several virulence factors, such as type 5a secreted autotransporter (T5aSSs) and membrane occupation and recognition nexus (MORN) domain-containing proteins, were upregulated under acidic conditions. A large number of proteins showed significant enrichments in multiple pathways involving fatty acid synthesis and butyrate synthesis. Proteomics analysis of the outer membrane vesicles secreted by pathogenic bacteria in the acidic tumor microenvironment is of great significance for elucidating the pathogenicity mechanism and its application in vaccine and drug delivery vehicles.

[Expression, purification, and characterization of the histidine kinase CarS from Fusobacterium nucleatum].

Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 ß-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarScyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarScyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarScyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarScyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.

High-Efficiency Reducing Strain for Producing Selenium Nanoparticles Isolated from Marine Sediment.

Selenium nanoparticles (SeNPs) are all important for research because they exhibit a higher degree of absorption and lower toxicity than that of their organic and inorganic forms. At present, there are few reports on marine strains that can reduce Se(IV) to generate Se(0). In this study, a strain that reduces sodium selenite to SeNPs with high efficiency was screened from 40 marine strains. The SeNPs-S produced by the whole cells and SeNPs-E produced by the extracellular extract were characterized by FTIR, UV, Raman, XRD and SEM. Based on the results, the two kinds of SeNPs exhibited obvious differences in morphology, and their surfaces were capped with different biomacromolecules. Due to the difference in shape and surface coating, opposite results were obtained for the antibacterial activity of SeNPs-S and SeNPs-E against Gram-positive and Gram-negative bacteria. Both SeNPs-S and SeNPs-E exhibited no obvious cytotoxicity at concentrations up to 100 µg/mL, but SeNPs-E retained lower cytotoxicity when its concentration increased to 200 µg/mL. This is the first report on the detailed difference between the SeNPs produced by whole cells and cell extracts.

Insights into the structure and function of the histidine kinase ComP from Bacillus amyloliquefaciens based on molecular modeling.

The ComPA two-component signal transduction system (TCS) is essential in Bacillus spp. However, the molecular mechanism of the histidine kinase ComP remains unclear. Here, we predicted the structure of ComP from Bacillus amyloliquefaciens Q-426 (BaComP) using an artificial intelligence approach, analyzed the structural characteristics based on the molecular docking results and compared homologous proteins, and then investigated the biochemical properties of BaComP. We obtained a truncated ComPS protein with high purity and correct folding in solution based on the predicted structures. The expression and purification of BaComP proteins suggested that the subdomains in the cytoplasmic region influenced the expression and stability of the recombinant proteins. ComPS is a bifunctional enzyme that exhibits the activity of both histidine kinase and phosphotransferase. We found that His571 played an obligatory role in the autophosphorylation of BaComP based on the analysis of the structures and mutagenesis studies. The molecular docking results suggested that the HATPase_c domain contained an ATP-binding pocket, and the ATP molecule was coordinated by eight conserved residues from the N, G1, and G2 boxes. Our study provides novel insight into the histidine kinase BaComP and its homologous proteins.

Expression, Purification, and Characterization of the Recombinant, Two-Component, Response Regulator ArlR from Fusobacterium nucleatum.

Fusobacterium nucleatum is associated with the incidence and development of multiple diseases, such as periodontitis and colorectal cancer (CRC). Until now, studies have proved only a few proteins to be associated with such pathogenic diseases. The two-component system is one of the most prevalent forms of bacterial signal transduction related to intestinal diseases. Here, we report a novel, recombinant, two-component, response regulator protein ArlR from the genome of F. nucleatum strain ATCC 25,586. We optimized the expression and purification conditions of ArlR; in addition, we characterized the interaction of this response regulator protein with the corresponding histidine kinase and DNA sequence. The full-length ArlR was successfully expressed in six E. coli host strains. However, optimum expression conditions of ArlR were present only in E. coli strain BL21 CodonPlus (DE3) RIL that was later induced with isopropyl ß-D-1-thiogalactopyranoside (IPTG) for 8 h at 25 °C. The SDS-PAGE analysis revealed the molecular weight of the recombinant protein as 27.3 kDa with approximately 90% purity after gel filtration chromatography. Because ArlR was biologically active after its purification, it accepted the corresponding phosphorylated histidine kinase phosphate group and bound to the analogous DNA sequence. The binding constant between ArlR and the corresponding histidine kinase was about 2.1 µM, whereas the binding constant between ArlR and its operon was 6.4 µM. Altogether, these results illustrate an effective expression and purification method for the novel two-component system protein ArlR.

Nuclear Respiratory Factor 1 (NRF-1) Controls the Activity Dependent Transcription of the GABA-A Receptor Beta 1 Subunit Gene in Neurons.

While the exact role of ß1 subunit-containing GABA-A receptors (GABARs) in brain function is not well understood, altered expression of the ß1 subunit gene (GABRB1) is associated with neurological and neuropsychiatric disorders. In particular, down-regulation of ß1 subunit levels is observed in brains of patients with epilepsy, autism, bipolar disorder and schizophrenia. A pathophysiological feature of these disease states is imbalance in energy metabolism and mitochondrial dysfunction. The transcription factor, nuclear respiratory factor 1 (NRF-1), has been shown to be a key mediator of genes involved in oxidative phosphorylation and mitochondrial biogenesis. Using a variety of molecular approaches (including mobility shift, promoter/reporter assays, and overexpression of dominant negative NRF-1), we now report that NRF-1 regulates transcription of GABRB1 and that its core promoter contains a conserved canonical NRF-1 element responsible for sequence specific binding and transcriptional activation. Our identification of GABRB1 as a new target for NRF-1 in neurons suggests that genes coding for inhibitory neurotransmission may be coupled to cellular metabolism. This is especially meaningful as binding of NRF-1 to its element is sensitive to the kind of epigenetic changes that occur in multiple disorders associated with altered brain inhibition.

Impact of apolipoprotein E (ApoE) polymorphism on brain ApoE levels.

Inheritance of the apoE4 allele (epsilon4) increases the risk of developing Alzheimer's disease; however, the mechanisms underlying this association remain elusive. Recent data suggest that inheritance of epsilon4 may lead to reduced apoE protein levels in the CNS. We therefore examined apoE protein levels in the brains, CSF and plasma of epsilon2/2, epsilon3/3, and epsilon4/4 targeted replacement mice. These apoE mice showed a genotype-dependent decrease in apoE levels; epsilon2/2 >epsilon3/3 >epsilon4/4. Next, we sought to examine the relative contributions of apoE4 and apoE3 in the epsilon3/4 mouse brains. ApoE4 represented 30-40% of the total apoE. Moreover, the absolute amount of apoE3 per allele was similar between epsilon3/3 and epsilon3/4 mice, implying that the reduced levels of total apoE in epsilon3/4 mice can be explained by the reduction in apoE4 levels. In culture medium from epsilon3/4 human astrocytoma or epsilon3/3, epsilon4/4 and epsilon3/4 primary astrocytes, apoE4 levels were consistently lower than apoE3. Secreted cholesterol levels were also lower from epsilon4/4 astrocytes. Pulse-chase experiments showed an enhanced degradation and reduced half-life of newly synthesized apoE4 compared with apoE3. Together, these data suggest that astrocytes preferentially degrade apoE4, leading to reduced apoE4 secretion and ultimately to reduced brain apoE levels. Moreover, the genotype-dependent decrease in CNS apoE levels, mirror the relative risk of developing AD, and suggest that low levels of total apoE exhibited by epsilon4 carriers may directly contribute to the disease progression, perhaps by reducing the capacity of apoE to promote synaptic repair and/or Abeta clearance.

Cellular dynamics of antisense oligonucleotides and short interfering RNAs.

We aim to compare quantitatively the dynamics of the effectiveness of antisense oligonucleotides (AS ODNs) versus short interfering RNAs (siRNAs) and relate their effectiveness to sequence metrics (e.g., predicted free energy of binding). AS ODNs against a quantitative model target, pd1EGFP (destabilized enhanced GFP [green fluorescent protein]), were selected using our thermodynamic model, and siRNA sequences were designed to be identical to the AS ODN sequences in the antisense strand. We evaluated d1EGFP inhibition in transiently and stably transfected Chinese hamster ovary (CHO) cells over time using flow cytometry. Overall, our results show that the rationally designed AS ODN and siRNA sequences proved effective inhibitors of GFP expression and suggest that certain regions of mRNA may be susceptible to both AS ODNs and siRNAs.

Multi-modal regulation of endogenous D1 dopamine receptor expression and function in the CAD catecholaminergic cell line.

Dopamine receptors exhibit tissue- and cell type-specific expression that is modulated during development, aging and in diseases such as Parkinson's. The molecular mechanisms regulating expression of dopamine receptors are not well understood, in part due to the lack of a model cell line that not only expresses endogenous dopamine receptors but also has the requisite regulatory mechanisms. Here, we demonstrate that the CAD catecholaminergic cell line expresses D1, D2, D3 and D5 dopamine receptor subtypes and associated signaling proteins. CAD cell differentiation induced by serum withdrawal increases the levels of D1 receptor mRNA by transcriptional up-regulation. This increase is also mimicked by the neurotrophin NT3. Interestingly, the increase of D1 receptor mRNA does not result in increased levels of D1 receptor protein in differentiated CAD cells. Furthermore, while the D1 receptor protein is expressed in differentiated CAD cells, it loses its ability to activate adenylyl cyclase. We demonstrate that the post-transcriptional regulation is not due to decreased D1 receptor mRNA stability or generation of a truncated D1 receptor mRNA, and that the down-regulation of D1 receptor function in differentiated CAD cells is mediated by post-translational mechanisms that decrease cell surface receptor expression by altering receptor processing and trafficking.