Identification and functional analysis of a galactosyltransferase capable of cholesterol glycolipid formation in the Lyme disease spirochete Borrelia burgdorferi

Borrelia burgdorferi (Bb), the etiological agent of Lyme disease, produces a series of simple glycolipids where diacylglycerol and cholesterol serve as the precursor. The cholesterol-based glycolipids, cholesteryl 6-O-acyl-β-D-galactopyranoside (ACGal) and cholesteryl-β-D-galactopyranoside (CGal) are immunogenic and proposed to contribute to the pathogenesis of Lyme disease. Detailed studies of CGal and ACGal in Bb have been hampered by a lack of knowledge of their underlying biosynthetic processes. The genome of Bb encodes four putative glycosyltransferases, and only one of these, BB0572, was predicted to be an inverting family 2 glycosyltransferase (GT2 enzyme) capable of using UDP-galactose as a substrate and forming a β-glycosidic bond. Comparison of the 42 kDa BB0572 amino acid sequence from Bb with other Borrelia spp demonstrates that this protein is highly conserved. To establish BB0572 as the galactosyltransferase capable of cholesterol glycolipid formation in Bb, the protein was produced as a recombinant product in Escherichia coli and tested in a cell-free assay with 14C-cholesterol and UDP-galactose as the substrates. This experiment resulted in a radiolabeled lipid that migrated with the cholesterol glycolipid standard of CGal when evaluated by thin layer chromatography. Additionally, mutation in the predicted active site of BB0572 resulted in a recombinant protein that was unable to catalyze the formation of the cholesterol glycolipid. These data characterize BB0572 as a putative cholesterol galactosyltransferase. This provides the first step in understanding how Bb cholesterol glycolipids are formed and will allow investigations into their involvement in pathogen transmission and disease development.

Introducing Novel Molecular-based Method for Quantification of Homologous Recombination Efficiency

BackgroundHomologous recombination (HR) pathway is a DNA double-stranded breaks repair pathway well-known for its high level of accuracy. Low HR pathway efficiency clinically known as homologous recombination deficiency (HRD) was identified in some cancers such as breast and ovarian cancers and studies have reported the sensitivity of HRD cancer cells to DNA repair inhibitors such as Olaparib. However, current techniques including immunofluorescence-based technique are qualitative-based, hence lack sensitivity to determine the functionality of HR pathway. Additionally, some of the techniques including gene expression arrays require expression study of wide range genes involve in HR pathway, which is not cost-effective. The aim of the study is to optimise a PCR-based assay (Norgen’s Homologous Recombination kit) that can be employed to quantitate HR efficiency in cells, which accurately reflects the functional status of HR pathway.Methods and FindingsThe kit has two test plasmids (dl-1 and dl-2) with partial deletions in the LacZ gene and the plasmids are generated from modification of pUC19. HR-proficient (HeLa and AsPC-1) and HR-deficient (CAPAN-1 cells) cancer cell lines were transfected with the two plasmids to generate functional LacZ gene (i.e. recombinant product). The recombinant product was quantified by real-time PCR. Although recombinant product was generated in all the cell lines, our real-time PCR demonstrated a high quantity of recombinant product in HeLa cell line whilst low quantity in CAPAN-1 and AsPC-1 cell lines. The quantity of recombinant product generated and quantified reflects HR pathway efficiency.ConclusionOverall, the results have provided some evidence that the PCR-based kit can be suitably employed for quantification of HR efficiency provided appropriate transfection method and reagent are used. However, further study is required to confirm HR efficiency status of AsPC-1 cells to ascertain the low HR efficiency detected by the kit in these cells.

Expression, Purification, and Verification of Recombinant Botulinum Neurotoxin Type A Binding Domain: A Comparison Between X33 and PichiaPink Strains of Pichia pastoris

Background: An effective method to develop a safe vaccine against botulism is to utilize molecular biology techniques to produce recombinant antigens, which provoke the immune response in the recipient organism. A suggested antigen is a specific recombinant fragment of the botulinum neurotoxin (BoNT), which elicits the predictable immune response and does not have any toxic effects. In this study, the binding domain of the heavy chain of BoNT serotype A, which is the responsible subunit for binding to the receptor(s) of presynaptic membranes in neuromuscular junctions, is the selected fragment of this toxin to be recombinantly produced. Objectives: In order to prevent a severe syndrome such as Botulism, developing efficient vaccines against it is a necessity. Efforts have been made to accomplish this throughout time; however, some have discontinued due to the risks and unreliability of their production and usage. Methods: The encoding gene of BoNT/A-Hc was cloned into two different strains of Pichia pastoris, which were compared to each other based on the yield of the recombinant product. Results: The results demonstrated that the expression of recombinant BoNT/A-Hc by PichiaPink strain was successful, and the achieved recombinant BoNT/A-Hc was subsequently purified and then verified by using the specific antibody and analytical methods. Conclusions: In contrast, the expression results from the X-33 strain were not significant.

Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes

Peptide mapping analysis is a regulatory expectation to verify the primary structure of a recombinant product sequence and to monitor post-translational modifications (PTMs). Although proteolytic digestion has been used for decades, it remains a labour-intensive procedure that can be challenging to accurately reproduce. Here, we describe a fast and reproducible protocol for protease digestion that is automated using immobilised trypsin on magnetic beads, which has been incorporated into an optimised peptide mapping workflow to show method transferability across laboratories. The complete workflow has the potential for use within a multi-attribute method (MAM) approach in drug development, production and QC laboratories. The sample preparation workflow is simple, ideally suited to inexperienced operators and has been extensively studied to show global applicability and robustness for mAbs by performing sample digestion and LC-MS analysis at four independent sites in Europe. LC-MS/MS along with database searching was used to characterise the protein and determine relevant product quality attributes (PQAs) for further testing. A list of relevant critical quality attributes (CQAs) was then established by creating a peptide workbook containing the specific mass-to-charge (m/z) ratios of the modified and unmodified peptides of the selected CQAs, to be monitored in a subsequent test using LC-MS analysis. Data is provided that shows robust digestion efficiency and low levels of protocol induced PTMs.

Fantastic yeasts and where to find them: the discovery of a predominantly clonal Cryptococcus deneoformans population in Saudi Arabian soils

ABSTRACT Cryptococcus deneoformans is an opportunist yeast pathogen and causative agent of meningoencephalitis in humans. It is known to be mainly distributed in temperate climates. Most of our current understanding of this species has come from clinical isolates, leaving environmental populations largely unexplored. The Middle East remains one such underexplored area with no published study to date investigating cryptococcal diversity in soil. In this study, we identified 76 C. deneoformans isolates from a survey of 562 soil samples collected from six cities in Saudi Arabia. Multilocus sequence typing revealed the presence of two major sequence types (STs), ST160 (n = 63) and ST294 (n = 9), along with four singleton STs, three of which were novel. One novel ST, ST613, was likely a recombinant product between ST160 and ST294. Among the 76 isolates, 75 belonged to mating type (MAT)α while one isolate was MATa. Our analyses suggest that the Saudi Arabian C. deneoformans population likely reproduces both asexually and sexually in nature. Our study is the first to report the occurrence of C. deneoformans in a desert climate, representing a novel expansion to this species’ currently known ecological niche.

Expression of spider toxin in entomopathogenic fungus Lecanicillium muscarium and selection of the strain showing efficient secretion of the recombinant protein

ABSTRACT Beta/delta-agatoxin-1 of spider Agelena orientalis was expressed in entomopathogenic fungus Lecanicillium muscarium. To ensure secretion of the recombinant product by the fungus, the signal secretory peptide of the Metarhizium anisopliae Mcl1 protein was inserted into the sequence. For detection of the recombinant product and selection of transformants, the toxin sequence was also fused with eGFP at the C-terminus. The gene encoding the A. orientalis toxin with the Mcl1 protein signal peptide was commercially synthesized, amplified and cloned into the vector pBARGPE1 designed for heterologous expression under the control of the PgpdA promoter and the trpC terminator of Aspergillus nidulans. A double selection on selective medium and microscopic analysis of transformants allowed obtaining a mitotically stable recombinant strain of L. muscarium. The recognition of the Mcl1 derived signal peptide in the cells of transformants and effective secretion of the hybrid product was confirmed by immunoblotting.