Investigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional Neisseria meningitidis serogroup W capsule polymerase

Abstract Objective Neisseria meningitidis is a Gram-negative bacterium that causes meningitis. N. meningitidis serogroup W (NmW) capsule polymerase synthesizes capsular polysaccharide of this serogroup. This enzyme could be a tool for meningococcal glycoconjugate vaccine development. Our long-term goal is to control activity of the NmW capsule polymerase for production of defined carbohydrates for vaccines. The enzyme lacks a simple, high-throughput activity assay. Here, we describe the use of high-throughput bioluminescence assays (CMP-Glo and UDP-Glo by Promega) to investigate NmW capsule polymerase activity. These assays detect free nucleotides produced during transfer of sugar from UDP-Galactose and CMP-Sialic Acid to an acceptor. Kinetic studies using NmW hydrolyzed polysaccharide (PS) acceptor are described as well as preliminary work with a sialic acid trimer (DP3) acceptor. Results In CMP-Glo kinetic studies, with constant donor (80 µM) and varied NmW hydrolyzed polysaccharide (0–2000 µg/mL), a Km of 629.2 ± 101.4 µg/mL and a Vmax of 0.8965 ± 0.05823 µM/min was obtained. Using UDP-Glo, Km and Vmax values of 13.84 ± 9.675 µM and 0.6205 ± 0.1331 µM/min were obtained with varied CMP-NeuNAc (0–80 µM) and constant acceptor (400 µg/mL) and UDP-Gal (80 µM). This is the first report of using bioluminescence assays for NmW kinetics.

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Structural determination of the capsular polysaccharide of Neisseria meningitidis group I: a two-dimensional NMR analysis

Biochemistry ◽

10.1021/bi00341a046 ◽

1985 ◽

Vol 24 (20) ◽

pp. 5592-5598 ◽

Cited By ~ 30

Author(s):

Francis Michon ◽

Jean Robert Brisson ◽

Rene Roy ◽

Fraser E. Ashton ◽

Harold J. Jennings

Keyword(s):

Neisseria Meningitidis ◽

Capsular Polysaccharide ◽

Structural Determination ◽

Two Dimensional ◽

Nmr Analysis ◽

Group I

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Evaluation of Recombinant Lipidated P2086 Protein as a Vaccine Candidate for Group B Neisseria meningitidis in a Murine Nasal Challenge Model

Infection and Immunity ◽

10.1128/iai.73.10.6838-6845.2005 ◽

2005 ◽

Vol 73 (10) ◽

pp. 6838-6845 ◽

Cited By ~ 23

Author(s):

Duzhang Zhu ◽

Ying Zhang ◽

Vicki Barniak ◽

Liesel Bernfield ◽

Alan Howell ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Capsular Polysaccharide ◽

Vaccine Development ◽

Vaccine Candidate ◽

Active Immunization ◽

Human Beings ◽

Nasal Tissue ◽

Potential Vaccine ◽

Group B ◽

Different Strains

ABSTRACT Neisseria meningitidis is a major causative agent of bacterial meningitis in human beings, especially among young children (≤2 years of age). Prevention of group B meningococcal disease represents a particularly difficult challenge in vaccine development, due to the inadequate immune response elicited against type B capsular polysaccharide. We have established an adult mouse intranasal challenge model for group B N. meningitidis to evaluate potential vaccine candidates through active immunization. Swiss Webster mice were inoculated intranasally with meningococci, and bacteria were recovered from the noses for at least 3 days postchallenge. Iron dextran was required in the bacterial inoculum to ensure sufficient meningococcal recovery from nasal tissue postchallenge. This model has been utilized to evaluate the potential of a recombinant lipidated group B meningococcal outer membrane protein P2086 (rLP2086) as a vaccine candidate. In this study, mice were immunized subcutaneously with purified rLP2086 formulated with or without an attenuated cholera toxin as an adjuvant. The mice were then challenged intranasally with N. meningitidis strain H355 or M982, and the colonization of nasal tissue was determined by quantitative culture 24 h postchallenge. We demonstrated that immunization with rLP2086 significantly reduced nasal colonization of mice challenged with the two different strains of group B N. meningitidis. Mice immunized with rLP2086 produced a strong systemic immunoglobulin G response, and the serum antibodies were cross-reactive with heterologous strains of group B N. meningitidis. The antibodies have functional activity against heterologous N. meningitidis strain, as demonstrated via bactericidal and infant rat protection assays. These results suggest that rLP2086 is a potential vaccine candidate for group B N. meningitidis.

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Synthesis and immunological evaluation of protein conjugates ofNeisseria meningitidisX capsular polysaccharide fragments

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.10.247 ◽

2014 ◽

Vol 10 ◽

pp. 2367-2376 ◽

Cited By ~ 20

Author(s):

Laura Morelli ◽

Damiano Cancogni ◽

Marta Tontini ◽

Alberto Nilo ◽

Sara Filippini ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Capsular Polysaccharide ◽

Vaccine Development ◽

Carrier Protein ◽

Immunological Activity ◽

Preclinical Stage ◽

Protein Conjugates ◽

Chemical Conjugation ◽

Immunological Evaluation

A vaccine to prevent infections from the emergingNeisseria meningitidisX (MenX) is becoming an urgent issue. Recently MenX capsular polysaccharide (CPS) fragments conjugated to CRM197as carrier protein have been confirmed at preclinical stage as promising candidates for vaccine development. However, more insights about the minimal epitope required for the immunological activity of MenX CPS are needed. We report herein the chemical conjugation of fully synthetic MenX CPS oligomers (monomer, dimer, and trimer) to CRM197. Moreover, improvements in some crucial steps leading to the synthesis of MenX CPS fragments are described. Following immunization with the obtained neoglycoconjugates, the conjugated trimer was demonstrated as the minimal fragment possessing immunogenic activity, even though significantly lower than a pentadecamer obtained from the native polymer and conjugated to the same protein. This finding suggests that oligomers longer than three repeating units are possibly needed to mimic the activity of the native polysaccharide.

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A High-Throughput Method for Enzyme Kinetic Studies

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057103257290 ◽

2003 ◽

Vol 8 (5) ◽

pp. 544-554 ◽

Cited By ~ 7

Author(s):

Lakshmi D. Saraswat ◽

Kimberley A. Caserta ◽

Kathy Laws ◽

Darren Wei ◽

Simon S. Jones ◽

...

Keyword(s):

Drug Metabolism ◽

High Throughput ◽

Kinetic Studies ◽

Metabolic Stability ◽

Metabolic Parameter ◽

High Throughput Method ◽

Multiple Samples ◽

Parameter Values

A simple and flexible setup for conducting drug metabolism studies is described in this report. A heating block was designed for the Multimek liquid handler platform for incubation of multiple samples at 37 °C in a 96-well format. This setup enables the rapid performance of drug metabolism experiments on a large number of samples. In this report, the authors present the validation of the system by 1) showing reproducible and consistent determination of the in vitro half-life of midazolam in every well across the entire plate and 2) determination of metabolic parameter values of midazolam, testosterone, diclofenac, warfarin, and dextromethorphan and inhibition parameter values of quinidine and ketoconazole, all comparable to literature values. In addition, the authors demonstrate the application of the setup to determining the metabolic stability of a set of proprietary compounds, the inhibition of activity of cytochrome P450 (CYP) enzymes, and the conduct of a single combination experiment that can simultaneously determine the metabolic stability and CYP inhibition activity. Overall, the system represents a simple, high-throughput and useful tool for drug metabolism screening in drug discovery. ( Journal of Biomolecular Screening 2003:544-554)

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Towards Computationally Guided Design and Engineering of a Neisseria Meningitidis Serogroup W Capsule Polymerase with Altered Substrate Specificity

10.20944/preprints202109.0339.v1 ◽

2021 ◽

Author(s):

Subhadra Paudel ◽

James Wachira ◽

Pumtiwitt C. McCarthy

Keyword(s):

Sialic Acid ◽

Metal Ions ◽

Neisseria Meningitidis ◽

Capsular Polysaccharide ◽

Heavy Metal Contamination ◽

Wastewater Treatment Plants ◽

Binding Capacity ◽

Health Concern ◽

Rational Approach ◽

Active Site Residues

Heavy metal contamination of drinking water is a public health concern that requires the development of more efficient bioremediation techniques. Absorption technologies, including biosorption, provide opportunities for improvements to increase the diversity of metal ions removed and overall binding capacity. Microorganisms are a key component in wastewater treatment plants and they naturally bind metal ions through surface macromolecules but with limited capacity. The long-term goal of this work is to engineer capsule polymerases to synthesize molecules with novel functionalities. In previously published work, we showed that the Neisseria meningitidis serogroup W (NmW) galactose-sialic acid (Gal—NeuNAc) heteropolysaccharide binds lead effectively, thereby demonstrating the potential for using this capsular polysaccharide in environmental decontamination applications. In this study, computational analysis of the NmW capsule polymerase galactosyltransferase (GT) domain was used to gain insight into how the enzyme could be modified to enable the synthesis N-acetylgalactosamine-sialic acid (GalNAc—NeuNAc) heteropolysaccharide. Various computational approaches, including molecular modeling with I-TASSER and molecular dynamics simulations (MD) with NAMD, were utilized to identify key amino acid residues in the substrate binding pocket of the GT domain that may be key to conferring UDP-GalNAc specificity. Through these combined strategies and using BshA, a UDP-GlcNAc transferase, as a structural template, several NmW active site residues were identified as mutational targets to accommodate the proposed N-acetyl group in UDP-GalNAc. Thus, a rational approach for potentially conferring new properties to bacterial capsular polysaccharides is demonstrated.

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