Antimicrobial Proteins for Human Health
Creators: name: Berhane, Nahom Ahferom
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2018
Subjects: Antibiotic resistance
protein purification
protein expression
refolding
biofilm
histone
OCX-32
eggshell

description: Bacteria are one of the largest causes of human disease, with millions of deaths every year attributed to bacterial infections, and they have become more difficult to tackle with the widespread emergence of antibiotic resistance. In this thesis, I describe my studies that pursued two approaches: one focus was on using antimicrobial histones as an alternative to treatment for antibiotic resistant bacteria; in another approach the recombinant version of an eggshell cuticle protein was expressed and purified for testing against food-safety pathogens. One major pathogen that is contributing to this challenge of antibiotic resistance is Staphylococcus aureus. The methicillin-resistant strain of S. aureus leads to increased hospital stays and increased mortality in patients. The impact of such pathogens is worsened when bacteria form surface-attached aggregates known as biofilms. Development of new approaches to eradicate antibiotic- resistant biofilms will benefit human health. This study looked at an alternative method to eradicate bacteria compared to traditional antibiotics. Histones with antimicrobial activity were extracted from chicken blood and tested against methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus biofilm (MSSA and MRSA). The histone mixture completely eradicated both strains in biofilm form at relatively low concentrations. In addition, the histone mixture also displayed fast kill kinetics against planktonic forms of the two strains. Finally, the interaction of the histone mixture with the bacterial membrane in MRSA biofilms was observed by scanning electron microscopy (SEM). Bacteria treated with the histone mixture showed clear morphological changes, including pore formation and cell collapse. Therefore, the histone mixture purified from chicken red blood cells could prove to be a good alternative to traditional antibiotics for protection against antibiotic-resistant strains of bacteria in their planktonic and biofilm forms. Reduction of food-borne illness is another important aspect in the promotion of human health. A significant contributor to food-borne illness is contaminated table eggs. The unfertilized egg can be contaminated by a variety of pathogens including Salmonella spp. and Bacillus spp. The egg is protected by the eggshell which is traversed by respiratory pores that are normally covered by a cuticle plug to restrict pathogen entry. This cuticle consists of several proteins including ovocaxlyin-32 (OCX-32). OCX-32 has a large number of naturally occurring haplotypes due non-synonymous single nucleotide polymorphisms (SNPs). In this study, the goal was to express five of the most common haplotypes of OCX-32 in Escherichia coli and purify the recombinant protein for assay of its antimicrobial activity. Five constructs that contain the cDNA of common OCX-32 haplotypes (A, B, C, D, and O) with a histidine tag at the C-terminus were generated. The constructs were subcloned into pGEX4T-1 vector which encodes Glutathione-S-transferase (GST) upstream of the multiple cloning site. My study developed methods to optimize the expression conditions, and to increase the solubility of the recombinant protein. Various expression strains of E. coli and solubility buffers were tested. In addition, the construct was subcloned into a plasmid containing the small ubiquitin-like modifier (SUMO) fusion tag; the solubility of the new SUMO-OCX-32 haplotype A recombinant fusion protein was evaluated. The best results were obtained by slow dialysis refolding of denatured SUMO-OCX-32 fusion protein. This recombinant protein showed almost complete solubility with minimal precipitation and was tested against the egg-related pathogen, Bacillus cereus. Unfortunately, the SUMO-OCX-32 recombinant protein did not inhibit growth of B. cereus. In my studies reported in this thesis, two very different approaches were taken. A histone mixture was isolated from an abundant starting material, which proved to be highly effective and promising in the eradication of S. aureus biofilms at relatively low concentrations. Alternatively, expression of a soluble recombinant protein for functional activity assay was very challenging and required the optimization of a number of methods to prepare soluble protein for testing. One of the methods tested proved effective in obtaining large amounts of soluble protein. However, further developmental work will be essential to determine if this approach is a viable strategy in acquiring functional protein.