Extraction of ADP-Heptose and Kdo2-Lipid A from E. coli Deficient in the Heptosyltransferase I Gene

Jozafina Milicaj,Nadiya Jaunbocus,Erika A Taylor,Colleen D Castro

doi:10.3390/app11188314

Jozafina Milicaj, Nadiya Jaunbocus + Show 2 more

Open Access

https://doi.org/10.3390/app11188314

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Journal: Applied sciences	Publication Date: Sep 8, 2021
Citations: 5	License type: CC BY 4.0

Affiliation: Wesleyan University

Abstract

The enzymes involved in lipopolysaccharide (LPS) biosynthesis, including Heptosyltransferase I (HepI), are critical for maintaining the integrity of the bacterial cell wall, and therefore these LPS biosynthetic enzymes are validated targets for drug discovery to treat Gram-negative bacterial infections. Enzymes involved in the biosynthesis of lipopolysaccharides (LPSs) utilize substrates that are synthetically complex, with numerous stereocenters and site-specific glycosylation patterns. Due to the relatively complex substrate structures, characterization of these enzymes has necessitated strategies to generate bacterial cells with gene disruptions to enable the extraction of these substrates from large scale bacterial growths. Like many LPS biosynthetic enzymes, Heptosyltransferase I binds two substrates: the sugar acceptor substrate, Kdo2-Lipid A, and the sugar donor substrate, ADP-l-glycero-d-manno-heptose (ADPH). HepI characterization experiments require copious amounts of Kdo2-Lipid A and ADPH, and unsuccessful extractions of these two substrates can lead to serious delays in collection of data. While there are papers and theses with protocols for extraction of these substrates, they are often missing small details essential to the success of the extraction. Herein detailed protocols are given for extraction of ADPH and Kdo2-Lipid A (KLA) from E. coli, which have had proven success in the Taylor lab. Key steps in the extraction of ADPH are clearing the extract through ultracentrifugation and keeping all water that touches anything in the extraction, including filters, at a pH of 8.0. Key steps in the extraction of KLA are properly lysing the dried down cells before starting the extraction, maximizing yield by allowing precipitate to form overnight, appropriately washing the pellet with phenol and dissolving the KLA in 1% TEA using visual cues, rather than a specific volume. These protocols led to increased yield and a higher success rate of extractions thereby enabling the characterization of HepI.

Highlights

Lipopolysaccharide (LPS) makes up approximately 30% of the outer membrane (Figure 1) of Gram-negative bacteria and is a multipurpose cellular component necessary for cellular motility, surface adhesion, and intermolecular interactions in a bacterial biofilm
LPS can be utilized by bacteria to prevent the cell permeation of hydrophobic molecules, thereby serving as a bacterial antibiotic resistance mechanism [4,5]
E. coli WBB06 cells were obtained from

Summary

Introduction

Lipopolysaccharide (LPS) makes up approximately 30% of the outer membrane (Figure 1) of Gram-negative bacteria and is a multipurpose cellular component necessary for cellular motility, surface adhesion, and intermolecular interactions in a bacterial biofilm. LPS is known for causing an immunological response in vivo, and is commonly referred to as an endotoxin [1,2,3]. LPS can be utilized by bacteria to prevent the cell permeation of hydrophobic molecules, thereby serving as a bacterial antibiotic resistance mechanism [4,5].

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