Suchergebnisse

Three novel glycosidases produced from Lactobacillus plantarum, so called Lp_0440, Lp_2777, and Lp_3525, were isolated and overexpressed on Escherichia coli containing a His-tag for specific purification. Their specific activity was evaluated against the hydrolysis of p-nitrophenylglycosides and p-nitrophenyl-6-phosphate glycosides (glucose and galactose) at pH 7. All three were modified with hyaluronic acid (HA) following two strategies: A simple coating by direct incubation at alkaline pH or direct chemical modification at pH 6.8 through preactivation of HA with carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at pH 4.8. The modifications exhibited important effect on enzyme activity and specificity against different glycopyranosides in the three cases. Physical modification showed a radical decrease in specific activity on all glycosidases, without any significant change in enzyme specificity toward monosaccharide (glucose or galactose) or glycoside (C-6 position free or phosphorylated). However, the surface covalent modification of the enzymes showed very interesting results. The glycosidase Lp_0440 showed low glycoside specificity at 25 °C, showing the same activity against p-nitrophenyl-glucopyranoside (pNP-Glu) or p-nitrophenyl-6-phosphate glucopyranoside (pNP-6P-Glu). However, the conjugated cHA-Lp_0440 showed a clear increase in the specificity towards the pNP-Glu and no activity against pNP-6P-Glu. The other two glycosidases (Lp_2777 and Lp_3525) showed high specificity towards pNP-6P-glycosides, especially to the glucose derivative. The HA covalent modification of Lp_3525 (cHA-Lp_3525) generated an enzyme completely specific against the pNP-6P-Glu (phosphoglycosidase) maintaining more than 80% of the activity after chemical modification. When the temperature was increased, an alteration of selectivity was observed. Lp_0440 and cHA-Lp_0440 only showed activity against p-nitrophenyl-galactopyranoside (pNP-Gal) at 40 °C, higher than at 25 °C in the case of the conjugated enzyme. ; This work was sponsored by the Spanish Government (Project No. AGL2017-84614-C2-2-R) and CSIC Project No. CSIC-PIE 201880E011). We also thank the Ministry of Education, Youth and Sports of the Community of Madrid and the European Social Fund for a contract to C.P.-R (PEJD-2017PRE/SAL-3762) in the program of Youth Employment and the Youth Employment Initiative (YEI) 2017. ; Peer reviewed

20 pags., 7 figs., 1 tab. ; Colorectal cancer pathogenesis and progression isassociated with the presence of Fusobacteriumnucleatum and the reduction of acetylated deriva-tives of spermidine, as well as dietary componentssuch as tannin-rich foods. We show that a new tan-nase orthologue of F. nucleatum (TanBFnn) has sig-nicant structural differences with its Lactobacillusplantarum counterpart affecting the ap covering theactive site and the accessibility of substrates. Crys-tallographic and molecular dynamics analysisrevealed binding of polyamines to a small cavity thatconnects the active site with the bulk solvent whichinteract with catalytically indispensable residues. Asa result, spermidine and its derivatives, particularlyN8-acetylated spermidine, inhibit the hydrolytic activ-ity of TanBFnnand increase the toxicity of gallotan-nins to F. nucleatum. Our results support a model inwhich the balance between the detoxicant activity ofTanBFnnand the presence of metabolic inhibitorscan dictate either conducive or unfavourable condi-tions for the survival of F. nucleatum. ; Supported by grants from the Spanish Ministry of Science and Innovation (MCI) cofinanced with FEDER funds (SAF2015‐65327‐R and RTI2018‐096494‐B‐100 to JA; AGL2017‐86757‐R to LA, SAF2015‐73549‐JIN to HR; RTI2018‐099592‐B‐C22 to GJO) and the Mizutani Foundation for Glycoscience (200077 to GJO). LA and GJO are supported by the Ramon y Cajal program (RYC‐2013‐13666 and RYC‐2013‐14706 respectively). JTC and AP are the recipients postdoctoral fellowships from the Basque Government. DB is the recipient of a MCI FPI fellowship. APC is the recipient of a fellowship from the University of the Basque Country. We thank the MCI for the Severo Ochoa Excellence accreditation (SEV‐2016‐0644) and the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs). JMM thanks the ALBA synchrotron for providing access time to the BL‐13 XALOC beamline. This work is supported by grants from the Jesús de Gangoiti Barrera Foundation ; Peer reviewed

Gut microbiota is a constant source of antigens and stimuli to which the resident immune system has developed tolerance. However, the mechanisms by which mononuclear phagocytes, specifically monocytes/macrophages, cope with these usually pro-inflammatory signals are poorly understood. Here, we show that innate immune memory promotes anti-inflammatory homeostasis, using as model strains of the commensal bacterium Lactiplantibacillus plantarum. Priming of monocytes/macrophages with bacteria, especially in its live form, enhances bacterial intracellular survival and decreases the release of pro-inflammatory signals to the environment, with lower production of TNF and higher levels of IL-10. Analysis of the transcriptomic landscape of these cells shows downregulation of pathways associated with the production of reactive oxygen species (ROS) and the release of cytokines, chemokines and antimicrobial peptides. Indeed, the induction of ROS prevents memory-induced bacterial survival. In addition, there is a dysregulation in gene expression of several metabolic pathways leading to decreased glycolytic and respiratory rates in memory cells. These data support commensal microbe-specific metabolic changes in innate immune memory cells that might contribute to homeostasis in the gut. ; Supported by grants from the Spanish Ministry of Science, Innovation and Universities (MCIU) co-financed with FEDER funds (RTI2018-096494-B-100 to JA; BFU2016-76872-R to EB; AGL2017-86757-R to LA; SAF2015-73549-JIN to HR; SAF2016–77433-R and PID2019-110240RB-I00 to RPR). AP is supported by a Postdoctoral Fellowship from the Basque Government. DB and TMM are recipients of MCIU FPI fellowships. APC is a recipient of a fellowship from the University of the Basque Country. LA and RPR are supported by the Ramon y Cajal program from the Spanish Ministry of Economy and Competitiveness. We thank the MCIU for the Severo Ochoa Excellence accreditation (SEV-2016-0644), the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs) and the Innovation Technology Department of the Bizkaia Province. This work was further supported by grants from the Jesús de Gangoiti Barrera Foundation. ; Peer reviewed