Biology subjects

Meyer, B. H.

Publications and source records attributed to Meyer, B. H..

5 recordsLinked to original sources

bioRxiv2025

Establishment of an efficient one-step enzymatic synthesis of cyclic-2,3-diphosphoglycerate

Extremolytes - unique compatible solutes produced by extremophiles - protect biological structures like membranes, proteins, and DNA under extreme conditions, including extremes of temperature and osmotic stress. These compounds hold significant potential for applications in pharmaceuticals, healthcare, cosmetics, and life sciences. However, despite their promise, only a few extremolytes, such as ectoine and hydroxyectoine, are commercially established, primarily due to the lack of efficient production strategies for other compounds. Cyclic 2,3-diphosphoglycerate (cDPG), a unique metabolite found in certain hyperthermophilic methanogenic Archaea, plays a key role in thermoprotection and is synthesized from 2-phosphoglycerate (2PG) through a two-step enzymatic process involving 2-phosphoglycerate kinase (2PGK) and cyclic-2,3-diphosphoglycerate synthetase (cDPGS). In this study, we present the development of an efficient in vitro enzymatic approach for the production of cDPG directly from 2,3-diphosphoglycerate (2,3DPG), leveraging the activity of the cDPGS from Methanothermus fervidus (MfcDPGS). We optimized the heterologous production of MfcDPGS in Escherichia coli by refining codon usage and expression conditions. The purification process was significantly streamlined through an optimized heat precipitation step, coupled with effective stabilization of MfcDPGS for both usage and storage by incorporating KCl, Mg2+, reducing agents and omission of an affinity tag. The recombinant MfcDPGS showed a Vmax of 38.2 U mg-1, with KM values of 1.52 mM for 2,3DPG and 0.55 mM for ATP. The enzyme efficiently catalyzed the complete conversion of 2,3DPG to cDPG. Remarkably, even at a scale of 100 mM, it achieved full conversion of 37.6 mg of 2,3DPG to cDPG within 180 minutes, using just 0.5 U of recombinant MfcDPGS at 55{degrees}C. These results highlight that MfcDPGS can be easily produced, rapidly purified, and sufficiently stabilized while delivering excellent conversion efficiency for cDPG synthesis as value-added product. Additionally, a kinetic model for MfcDPGS activity was developed, providing a crucial tool to simulate and scale up cDPG production for industrial applications. This streamlined process offers significant advantages for the scalable synthesis of cDPG, paving the way for further biochemical and industrial applications of this extremolyte.

bioengineering↗

bioRxiv2024

Rhamnose Polysaccharide-Decorated Outer Membrane Vesicles as a Vaccine Candidate Targeting Group A Streptococcus from Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

Group A Streptococcus (Strep A) cause a wide range of human-exclusive infections, annually killing more than 500,000 people. Antibiotic resistance incidence of invasive Strep A tripled in the past decade and emphasises the need to develop a universal Strep A vaccine. In this study, we developed recombinant rhamnose polysaccharides (RhaPS), a validated universal Strep A vaccine candidate, presented on E. coli outer membrane vesicles (OMVs). We investigated OMV-RhaPS for their immunogenicity in the mouse and rabbit models. Through flow cytometry, ELISA, Luminex assays, and immunofluorescence microscopy, we demonstrated that RhaPS-specific antibodies recognise Strep A strains via the Group A Carbohydrate (GAC) in S. pyogenes and the newly emerged S. dysgalactiae subsp. equisimilis. Elevated IL-17a levels from RhaPS-OMV-immunised splenocytes indicate the RhaPS-specific stimulation of long-term memory immune cells. We are the first to report the efficacy and potency of recombinant produced RhaPS inducing humoral-mediated immune responses and triggering antibodies that recognise Strep A bacteria.

microbiology↗

bioRxiv2021

Recombinant Group A Carbohydrate backbone embedded into Outer Membrane Vesicles is a potent vaccine candidate targeting Group A Streptococcus from Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

BackgroundGroup A Streptococcus (GAS) are responsible for a wide range of human-exclusive infections, annually killing more than 500,000 people. Antibiotic resistance incidence of invasive GAS tripled in the past decade and emphasises the need to develop a universal GAS vaccine. We have produced, for the first time, a recombinant polyrhamnose backbone (pRha), a validated universal GAS vaccine candidate. E. coli outer membrane vesicles (OMVs) carrying pRha were investigated for their immunogenicity and efficacy in an animal model. MethodsOMVs decorated with pRha were administered to C57BL/6J mouse and rabbit models. Flow cytometry, ELISA, Luminex, immunofluorescence microscopy and serum bactericidal assay assays were conducted to investigate the ability of pRha-specific antibodies to recognise and kill clinical (hypervirulent) GAS strains. ResultsOur results suggest that pRha-OMVs induce specific antibodies which recognise Group A Carbohydrate (GAC) from S pyogenes and S. dysgalactiae subsp. equisimilis. Increased IgG levels correlate with increased bactericidal killing of the hypervirulent GAS M89 strain. Elevated IL-17a from pRha-OMV-immunised splenocytes indicates possible stimulation of long-term memory immune cells. ConclusionWe are the first to report efficacy and potency of this unique, exogenously produced polysaccharide, pRha, in the induction of humoral-mediated immune responses to GAS. TopicStreptococcus pyogenes, immunoglobulins, polysaccharides, opsonophagocytosis, acute rheumatic fever, M protein, invasive Group A Streptococcus, hyaluronic acid

microbiology↗

bioRxiv2021

N-glycan core synthesis by Alg24 strengthens the hypothesis of an archaeal origin of eukaryal N-glycosylation

Protein N-glycosylation is the most common posttranslational modifications found in all three domains of life. The crenarchaeal N-glycosylation begins with the synthesis of a lipid-linked chitobiose core structure, identical to that in eukaryotes. Here, we report the identification of a thermostable archaeal beta-1,4-N-acetylglucosaminyltransferase, named archaeal glycosylation enzyme 24 (Agl24), responsible for the synthesis of the N-glycan chitobiose core. Biochemical characterization confirmed the function as an inverting {beta}-D-GlcNAc-(1[->]4)--D-GlcNAc-diphosphodolichol glycosyltransferase. Substitution of a conserved histidine residue, found also in the eukaryotic and bacterial homologs, demonstrated its functional importance for Agl24. Furthermore, bioinformatics and structural modeling revealed strong similarities between Agl24 and both the eukaryotic Alg14/13 and a distant relation to the bacterial MurG, which catalyze the identical or a similar process, respectively. Our data, complemented by phylogenetic analysis of Alg13 and Alg14, revealed similar sequences in Asgardarchaeota, further supporting the hypothesis that the Alg13/14 homologs in eukaryotes have been acquired during eukaryogenesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/427365v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@13d1ffdorg.highwire.dtl.DTLVardef@1ff48d0org.highwire.dtl.DTLVardef@1056e75org.highwire.dtl.DTLVardef@196ff58_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFirst identification and characterization of a thermostable {beta}-D-GlcNAc-(1[->]4)--D-GlcNAc-diphosphodolichol glycosyltransferase (GT family 28) in Archaea. C_LIO_LIA highly conserved histidine, within a GGH motif in Agl24, Alg14, and MurG, is essential for function of Agl24. C_LIO_LIAgl24-like homologs are broadly distributed among Archaea. C_LIO_LIThe eukaryotic Alg13 and Alg14 are closely related to the Asgard homologs, suggesting their acquisition during eukaryogenesis. C_LI

microbiology↗

bioRxiv2018

Identification of lead compounds targeting the dTDP-L-rhamnose biosynthesis pathway using Streptococcus pyogenes

Biosynthesis of the nucleotide sugar precursor dTDP-L-rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS) and Streptococcus mutans. Those pathogens require dTDP-L-rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirm that GAS RmlB and RmlC are critical for dTDP-L-rhamnose biosynthesis through their action as dTDP-glucose-4,6-dehydratase and dTDP-4-keto-6-deoxyglucose-3,5-epimerase enzymes, respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues in these enzymes. Bio-layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP-rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS as well as several other rhamnose-dependent streptococcal pathogens with an MIC50 of 120-410 M. We therefore conclude that inhibition of dTDP-L-rhamnose biosynthesis such as Ri03 affect streptococcal viability and can serve as a lead compound for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria.

microbiology↗