Genes Involved in the Endoplasmic Reticulum N-Glycosylation Pathway of the Red Microalga Porphyridium sp.: A Bioinformatic Study

N-glycosylation is one of the most important post-translational modifications that influence protein polymorphism, including protein structures and their functions. Although this important biological process has been extensively studied in mammals, only limited knowledge exists regarding glycosylation in algae. The current research is focused on the red microalga Porphyridium sp., which is a potentially valuable source for various applications, such as skin therapy, food, and pharmaceuticals. The enzymes involved in the biosynthesis and processing of N-glycans remain undefined in this species, and the mechanism(s) of their genetic regulation is completely unknown. In this study, we describe our pioneering attempt to understand the endoplasmic reticulum N-Glycosylation pathway in Porphyridium sp., using a bioinformatic approach. Homology searches, based on sequence similarities with genes encoding proteins involved in the ER N-glycosylation pathway (including their conserved parts) were conducted using the TBLASTN function on the algae DNA scaffold contigs database. This approach led to the identification of 24 encoded-genes implicated with the ER N-glycosylation pathway in Porphyridium sp. Homologs were found for almost all known N-glycosylation protein sequences in the ER pathway of Porphyridium sp.; thus, suggesting that the ER-pathway is conserved; as it is in other organisms (animals, plants, yeasts, etc.).     

Novel (+)-Neoisopulegol-Based O-Benzyl Derivatives as Antimicrobial Agents

Discovery of novel antibacterial agents with new structures, which combat pathogens is an urgent task. In this study, a new library of (+)-neoisopulegol-based O-benzyl derivatives of aminodiols and aminotriols was designed and synthesized, and their antimicrobial activity against different bacterial and fungal strains were evaluated. The results showed that this new series of synthetic O-benzyl compounds exhibit potent antimicrobial activity. Di-O-benzyl derivatives showed high activity against Gram-positive bacteria and fungi, but moderate activity against Gram-negative bacteria. Therefore, these compounds may serve a good basis for antibacterial and antifungal drug discovery. Structure–activity relationships were also studied from the aspects of stereochemistry of the O-benzyl group on cyclohexane ring and the substituent effects on the ring system.     

Monitoring of Intracellular Tau Aggregation Regulated by OGA/OGT Inhibitors

Abnormal phosphorylation of tau has been considered as a key pathogenic mechanism inducing tau aggregation in multiple neurodegenerative disorders, collectively called tauopathies. Recent evidence showed that tau phosphorylation sites are protected with O-linked β-N-acetylglucosamine (O-GlcNAc) in normal brain. In pathological condition, tau is de-glycosylated and becomes a substrate for kinases. Despite the importance of O-GlcNAcylation in tau pathology, O-GlcNAc transferase (OGT), and an enzyme catalyzing O-GlcNAc to tau, has not been carefully investigated in the context of tau aggregation. Here, we investigated intracellular tau aggregation regulated by BZX2, an inhibitor of OGT. Upon the inhibition of OGT, tau phosphorylation increased 2.0-fold at Ser199 and 1.5-fold at Ser396, resulting in increased tau aggregation. Moreover, the BZX2 induced tau aggregation was efficiently reduced by the treatment of Thiamet G, an inhibitor of O-GlcNAcase (OGA). Our results demonstrated the protective role of OGT in tau aggregation and also suggest the counter-regulatory mechanism of OGA and OGT in tau pathology.     

Catalysis and deactivation of montmorillonite K10 in the aryl <Emphasis Type="Italic">O</Emphasis>-glycosylation of glycosyl trichloroacetoimidates

The catalysis of montmorillonite K10 (MK10) for aryl O-glycosylation of glycosyl trichloroacetimidates was investigated. It was found that the catalyst MK10 is deactivated gradually in the recycle glycosylation. The fresh and the deactivated catalysts were characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Thermogravimetric analysis (TGA), and N₂ adsorption-desorption. The results show that the eliminated trichloroacetamide molecule deposits on the MK10, which blocks and poisons the active sites, resulting in the deactivation of the catalyst. The regeneration of the deactivated MK10 by calcination was studied preliminarily.     

Variation,Modification and Engineering of Lipid A in Endotoxin of Gram-Negative Bacteria

Lipid A of Gram-negative bacteria is known to represent a central role for the immunological activity of endotoxin. Chemical structure and biosynthetic pathways as well as specific receptors on phagocytic cells had been clarified by the beginning of the 21st century. Although the lipid A of enterobacteria including Escherichia coli share a common structure, other Gram-negative bacteria belonging to various classes of the phylum Proteobacteria and other taxonomical groups show wide variety of lipid A structure with relatively decreased endotoxic activity compared to that of E. coli. The structural diversity is produced from the difference of chain length of 3-hydroxy fatty acids and non-hydroxy fatty acids linked to their hydroxyl groups. In some bacteria, glucosamine in the backbone is substituted by another amino sugar, or phosphate groups bound to the backbone are modified. The variation of structure is also introduced by the enzymes that can modify electrostatic charges or acylation profiles of lipid A during or after its synthesis. Furthermore, lipid A structure can be artificially modified or engineered by the disruption and introduction of biosynthetic genes especially those of acyltransferases. These technologies may produce novel vaccine adjuvants or antagonistic drugs derived from endotoxin in the future.     

Can Gram-Negative Bacteria Develop Resistance to Antimicrobial Blue Light Treatment?

Antimicrobial blue light (aBL) treatment is considered low risk for the development of bacterial resistance and tolerance due to its multitarget mode of action. The aim of the current study was to demonstrate whether tolerance development occurs in Gram-negative bacteria. We evaluated the potential of tolerance/resistance development in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa and demonstrated that representative Gram-negative bacteria may develop tolerance to aBL. The observed adaption was a stable feature. Assays involving E. coli K-12 tolC-, tolA-, umuD-, and recA-deficient mutants revealed some possible mechanisms for aBL tolerance development.     

Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)

Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets.     

Graphene nanoplatelets/Cr2O3 nanocomposites as novel nanoantibiotics: Towards control of multiple drug resistant bacteria

Drug resistance resulting due to the abuse of antibiotics can possibly be fatal for human beings. It, therefore, is required to develop novel nanoantibiotics to fight with the bacterial infections. In this work, we report graphene nanoplatelets/Cr₂O₃ nanocomposites (GNPs/Cr₂O₃) as a potential nanomedicine. Antibacterial characteristics of GNPs/Cr₂O₃ nanocomposites have been investigated against Pseudomonas aeruginosa and Staphylococcus aureus. GCr₂O₃-II with the optimized GNPs’ content shows excellent antibacterial performance with 84.25% growth inhibition of S. aureus (Gram-positive bacteria) and 80.76% growth inhibition of -P. aeruginosa (Gram-negative bacteria). This can be attributed to the synergistic contribution of Cr₂O₃ nanoflakes and GNPs/Cr₂O₃ nanocomposites, towards bacterial membrane disruption, that may be caused by the sharp edges of GNPs and induction of the oxidative stress by Cr₂O₃ nanoflakes. Therefore, this study suggests that GNPs/Cr₂O₃ nanocomposites can be employed as an innovative nanoantibiotics for pathogen control.     

Midecamycin Is Inactivated by Several Different Sugar Moieties at Its Inactivation Site

Glycosylation inactivation is one of the important macrolide resistance mechanisms. The accumulated evidences attributed glycosylation inactivation to a glucosylation modification at the inactivation sites of macrolides. Whether other glycosylation modifications lead to macrolides inactivation is unclear. Herein, we demonstrated that varied glycosylation modifications could cause inactivation of midecamycin, a 16-membered macrolide antibiotic used clinically and agriculturally. Specifically, an actinomycetic glycosyltransferase (GT) OleD was selected for its glycodiversification capacity towards midecamycin. OleD was demonstrated to recognize UDP-D-glucose, UDP-D-xylose, UDP-galactose, UDP-rhamnose and UDP-N-acetylglucosamine to yield corresponding midecamycin 2′-O-glycosides, most of which displayed low yields. Protein engineering of OleD was thus performed to improve its conversions towards sugar donors. Q327F was the most favorable variant with seven times the conversion enhancement towards UDP-N-acetylglucosamine. Likewise, Q327A exhibited 30% conversion enhancement towards UDP-D-xylose. Potent biocatalysts for midecamycin glycosylation were thus obtained through protein engineering. Wild OleD, Q327F and Q327A were used as biocatalysts for scale-up preparation of midecamycin 2′-O-glucopyranoside, midecamycin 2′-O-GlcNAc and midecamycin 2′-O-xylopyranoside. In contrast to midecamycin, these midecamycin 2′-O-glycosides displayed no antimicrobial activities. These evidences suggested that besides glucosylation, other glycosylation patterns also could inactivate midecamycin, providing a new inactivation mechanism for midecamycin resistance. Cumulatively, glycosylation inactivation of midecamycin was independent of the type of attached sugar moieties at its inactivation site.     

MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.     

Novel Quinic Acid Glycerates from Tussilago farfara Inhibit Polypeptide GalNAc-Transferase

The discovery of a bioactive inhibitor tool for human polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts), the initiating enzyme for mucin-type O-glycosylation, remains challenging. In the present study, we identified an array of quinic acid derivatives, including four new glycerates ( 1 – 4 ) from Tussilago farfara, a traditional Chinese medicinal plant, as active inhibitors of GalNAc-T2 using a combined screening approach with a cell-based T2-specific sensor and purified enzyme assay. These inhibitors dose-dependently inhibited human GalNAc-T2 but did not affect O-linked N-acetylglucosamine transferase (OGT), the other type of glycosyltransferase. Importantly, they are not cytotoxic and retain inhibitory activity in cells lacking elongated O-glycans, which are eliminated by the CRISPR/Cas9 gene editing tool. A structure-activity relationship study unveiled a novel quinic acid-caffeic acid conjugate pharmacophore that directs inhibition. Overall, these new natural product inhibitors could serve as a basis for developing an inhibitor tool for GalNAc-T2.     

Fosfomycin Resistance Evolutionary Pathways of Stenotrophomonas maltophilia in Different Growing Conditions

The rise of multidrug-resistant Gram-negative pathogens and the lack of novel antibiotics to address this problem has led to the rescue of old antibiotics without a relevant use, such as fosfomycin. Stenotrophomonas maltophilia is a Gram-negative, non-fermenter opportunistic pathogen that presents a characteristic low susceptibility to several antibiotics of common use. Previous work has shown that while the so-far described mechanisms of fosfomycin resistance in most bacteria consist of the inactivation of the target or the transporters of this antibiotic, as well as the production of antibiotic-inactivating enzymes, these mechanisms are not selected in S. maltophilia fosfomycin-resistant mutants. In this microorganism, fosfomycin resistance is caused by the inactivation of enzymes belonging to its central carbon metabolism, hence linking metabolism with antibiotic resistance. Consequently, it is relevant to determine how different growing conditions, including urine and synthetic sputum medium that resemble infection, could impact the evolutionary pathways towards fosfomycin resistance in S. maltophilia. Our results show that S. maltophilia is able to acquire high-level fosfomycin resistance under all tested conditions. However, although some of the genetic changes leading to resistance are common, there are specific mutations that are selected under each of the tested conditions. These results indicate that the pathways of S. maltophilia evolution can vary depending on the infection point and provide information for understanding in more detail the routes of fosfomycin resistance evolution in S. maltophilia.     

Catalysis and deactivation of montmorillonite K10 in the aryl O-glycosylation of glycosyl trichloroacetoimidates

The catalysis of montmorillonite K10 (MK10) for aryl O-glycosylation of glycosyl trichloroacetimidates was investigated. It was found that the catalyst MK10 is deactivated gradually in the recycle glycosylation. The fresh and the deactivated catalysts were characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Thermogravimetric analysis (TGA), and N₂ adsorption-desorption. The results show that the eliminated trichloroacetamide molecule deposits on the MK10, which blocks and poisons the active sites, resulting in the deactivation of the catalyst. The regeneration of the deactivated MK10 by calcination was studied preliminarily.     

Antibacterial activity and corrosion resistance of Ta2O5 thin film and electrospun PCL/MgO-Ag nanofiber coatings on biodegradable Mg alloy implants

Biodegradable magnesium (Mg) alloys have drawn considerable attention for use in orthopedic implants, but their antibacterial activity and corrosion resistance still require improvement. In the present work, functional Ta₂O₅ (tantalum pentoxide) compact layers and PCL/MgO-Ag (poly (ε-caprolactone)/magnesium oxide-silver) nanofiber porous layers were subsequently deposited on Mg alloys via reactive magnetron sputtering and electrospinning, respectively, to improve anticorrosion and antibacterial performance. Sputter coating of the Ta₂O₅ resulted in a thick layer (～1?μm) with an amorphous structure and high adhesive strength. The nanostructure exhibited bubble-like patterns with no obvious nano-cracks, nano-porosities, or pinholes. The electrospun PCL/MgO-Ag nanofiber coating was porous, smooth, and plain with no obvious beads. In vitro corrosion tests demonstrated the PCL/MgO-Ag nanofiber-coated alloy had greater corrosion resistance than a Ta₂O₅ sputter-coated alloy or uncoated Mg alloy. The additional electrospun PCL/MgO-Ag nanofiber coating also had greater antibacterial behavior toward Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria than the Ta₂O₅-coated or uncoated alloy specimens. Increasing the MgO-Ag concentration of the nanofibers from 1 to 3?wt% increased antibacterial activity. The combination of Ta₂O₅ and PCL/MgO-Ag nanofiber coatings on Mg alloys may therefore have potential applications for reducing bone infection as related to orthopedic implants for bone repair.     

Electron transfer pathways in microbial oxygen biocathodes

The ability of some bacteria to enhance the rate of cathodic oxygen reduction to water has been recently discovered, opening the way to an entirely renewable and environmentally friendly concept of biocathode. In this study we reveal that several mechanisms may induce catalytic effects by bacteria. These comprise mechanisms that are putatively beneficial to the bacteria as well as mechanisms which are merely side effects, including quinone autoxidation and direct O₂ reduction by heme compounds. Here we showed that 1 μM of ACNQ is able to generate a significant catalytic wave for oxygen reduction, with onset at approximately 0 V vs. SHE. Similarly, adsorption of hemin on a carbon surface catalyses O₂ reduction to H₂O₂ with an onset of +0.2 V vs. SHE. To evaluate the catalytic pathways of live cells on cathodic oxygen reduction, two species of electrochemically active bacteria were selected as pure cultures, namely Acinetobacter calcoaceticus and Shewanella putrefaciens. The former appears to exploit a self-excreted redox compound with redox characteristics matching those of pyrroloquinoline quinone (PQQ) for extracellular electron transfer. The latter appears to utilise outer membrane-bound redox compounds. Interaction of quinones and cytochromes with the membrane-bound electron transfer chain is yet to be proven.     

Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efficacy,Synergy, Resistance and Mechanistic Studies

Antimicrobial resistant (AMR) bacteria constitute a global health concern. Helicobacter pylori is a Gram-negative bacterium that infects about half of the human population and is a major cause of peptic ulcer disease and gastric cancer. Increasing resistance to triple and quadruple H. pylori eradication therapies poses great challenges and urges the development of novel, ideally narrow spectrum, antimicrobials targeting H. pylori. Here, we describe the antimicrobial spectrum of a family of nitrobenzoxadiazol-based antimicrobials initially discovered as inhibitors of flavodoxin: an essential H. pylori protein. Two groups of inhibitors are described. One group is formed by narrow-spectrum compounds, highly specific for H. pylori, but ineffective against enterohepatic Helicobacter species and other Gram-negative or Gram-positive bacteria. The second group includes extended-spectrum antimicrobials additionally targeting Gram-positive bacteria, the Gram-negative Campylobacter jejuni, and most Helicobacter species, but not affecting other Gram-negative pathogens. To identify the binding site of the inhibitors in the flavodoxin structure, several H. pylori-flavodoxin variants have been engineered and tested using isothermal titration calorimetry. An initial study of the inhibitors capacity to generate resistances and of their synergism with antimicrobials commonly used in H. pylori eradication therapies is described. The narrow-spectrum inhibitors, which are expected to affect the microbiota less dramatically than current antimicrobial drugs, offer an opportunity to develop new and specific H. pylori eradication combinations to deal with AMR in H. pylori. On the other hand, the extended-spectrum inhibitors constitute a new family of promising antimicrobials, with a potential use against AMR Gram-positive bacterial pathogens.     

Studies on Properties of Rice Straw/Polymer Nanocomposites Based on Polycaprolactone and Fe3O4 Nanoparticles and Evaluation of Antibacterial Activity

Modified rice straw/Fe₃O₄/polycaprolactone nanocomposites (ORS/Fe₃O₄/PCL-NCs) have been prepared for the first time using a solution casting method. The RS/Fe₃O₄-NCs were modified with octadecylamine (ODA) as an organic modifier. The prepared NCs were characterized by using X-ray powder diffraction (XRD), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Thermogravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FT-IR). The XRD results showed that as the intensity of the peaks decreased with the increase of ORS/Fe₃O₄-NCs content in comparison with PCL peaks, the Fe₃O₄-NPs peaks increased from 1.0 to 60.0 wt. %. The TEM and SEM results showed a good dispersion of ORS/Fe₃O₄-NCs in the PCL matrix and the spherical shape of the NPs. The TGA analysis indicated thermal stability of ORS/Fe₃O₄-NCs increased after incorporation with PCL but the thermal stability of ORS/Fe₃O₄/PCL-NCs decreased with the increase of ORS/Fe₃O₄-NCs content. Tensile strength was improved with the addition of 5.0 wt. % of ORS/Fe₃O₄-NCs. The antibacterial activities of the ORS/Fe₃O₄/PCL-NC films were examined against Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) by diffusion method using nutrient agar. The results indicated that ORS/Fe₃O₄/PCL-NC films possessed a strong antibacterial activity with the increase in the percentage of ORS/Fe₃O₄-NCs in the PCL.     

Plant Defensins NaD1 and NaD2 Induce Different Stress Response Pathways in Fungi

Nicotiana alata defensins 1 and 2 (NaD1 and NaD2) are plant defensins from the ornamental tobacco that have antifungal activity against a variety of fungal pathogens. Some plant defensins interact with fungal cell wall O-glycosylated proteins. Therefore, we investigated if this was the case for NaD1 and NaD2, by assessing the sensitivity of the three Aspergillus nidulans (An) O-mannosyltransferase (pmt) knockout (KO) mutants (An∆pmtA, An∆pmtB, and An∆pmtC). An∆pmtA was resistant to both defensins, while An∆pmtC was resistant to NaD2 only, suggesting NaD1 and NaD2 are unlikely to have a general interaction with O-linked side chains. Further evidence of this difference in the antifungal mechanism was provided by the dissimilarity of the NaD1 and NaD2 sensitivities of the Fusarium oxysporum f. sp. lycopersici (Fol) signalling knockout mutants from the cell wall integrity (CWI) and high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathways. HOG pathway mutants were sensitive to both NaD1 and NaD2, while CWI pathway mutants only displayed sensitivity to NaD2.