Tissue homeostasis is necessary for optimal organ functioning. The onset of tissue trauma compromises the homeostatic environment resulting in widespread cell death with the likelihood of exposure to invading micro-organisms. Early stage elimination of microbes and immunomodulation is co-ordinated by leukocytes of the innate immune system of which neutrophils and macrophages play a pivotal role. Leukocyte-released pro-inflammatory factors are vital in the containment of infection but bring with it a degree of collateral tissue destruction. Thus cascading stages during inflammation must be tightly regulated to bring about timely tissue regeneration and regained homeostasis. However, chronic inflammatory diseases e.g. rheumatoid arthritis highlights the existence of defective regulation at numerous stages during this transition, often leading to debilitating disease progression. Recently published findings by our research group identified the anti-inflammatory properties of α-defensin - an anti-microbial peptide released from dying human neutrophils - on stimulated macrophages. Thus the main objective of my research was to gain an understanding into the molecular actions of α-defensins which inhibit the macrophage inflammatory potential. Strong evidence supported the propensity of α-defensins to inhibit both intracellular and secreted protein synthesis, as assessed by de novo 35S-radiolabeled Methionine incorporation. Inhibition was not attributed to endoplasmic reticulum stress events, a common diagnosis in the regulation of global translation. Supporting evidence using cell-free systems identified a fundamental block in translation with the inclusion of α-defensin. Biochemical studies linked the ability of α-defensin to bind non-specifically to oligonucleotide sequences. This binding potential was also demonstrated on ribosomal RNA (rRNA), impeding its migration through electrophoretic gels. Immunocytochemical assays proposed an emerging suggestion of α-defensins in macrophages concentrated in close proximity to ribosomes around the perinuclear region. Evidence of suggested defensin/ribosome accumulation after 24hrs after treatment were attempted but to date remained unconfirmed. Attempts to determine the fate of these proposed accumulations were inconclusive, assessed by autophagy assays and ribosome semiquantitation. This thesis describes for the first time an enhanced understanding into the intracellular inhibitory mechanisms of α-defensins on macrophages and possibly other cell types. Understanding the molecular impact of α-defensins provide key insights into this novel inflammatory regulator, with the potential to be utilized in future immunotherapies.

INTRODUCTION: Platelets are involved in the inflammatory and thrombotic complications associated with atherosclerosis. Human neutrophil peptides (HNP), released from activated neutrophils, demonstrate inflammatory effects related to lesion development. HNP bind the low-density lipoprotein receptor (LR) family member LRP1 and LRP8 is the only member on platelets. HYPOTHESIS: HNP enhance platelet activation and aggregation through interactions with LRP8. METHODS: Platelet activation and aggregation in response to HNP were determined using flow cytometry and aggregometry. Activation was also examined in the presence of recombinant LRP8 and in LRP8 knockout platelets. RESULTS: HNP activate platelets as determined by P-selectin expression and the formation of microparticles. HNP sensitize platelets enhancing their aggregatory response to ADP. Lastly, LRP8 plays a role in HNP-induced platelet activation. CONCLUSIONS: With an improved understanding of the mechanism by which HNP induce platelet activation, we may be able to devise therapeutic strategies to treat patients with cardiovascular diseases.

INTRODUCTION: Platelets are involved in the inflammatory and thrombotic complications associated with atherosclerosis. Human neutrophil peptides (HNP), released from activated neutrophils, demonstrate inflammatory effects related to lesion development. HNP bind the low-density lipoprotein receptor (LR) family member LRP1 and LRP8 is the only member on platelets. HYPOTHESIS: HNP enhance platelet activation and aggregation through interactions with LRP8. METHODS: Platelet activation and aggregation in response to HNP were determined using flow cytometry and aggregometry. Activation was also examined in the presence of recombinant LRP8 and in LRP8 knockout platelets. RESULTS: HNP activate platelets as determined by P-selectin expression and the formation of microparticles. HNP sensitize platelets enhancing their aggregatory response to ADP. Lastly, LRP8 plays a role in HNP-induced platelet activation. CONCLUSIONS: With an improved understanding of the mechanism by which HNP induce platelet activation, we may be able to devise therapeutic strategies to treat patients with cardiovascular diseases.

β-defensins are a class of mammalian defence peptides with therapeutic potential because of their ability to kill bacteria and attract host immune cells. In order to realise this potential, it is necessary to understand how the functions of these peptides are related to their structures. This thesis presents biophysical analysis of β- defensins and related peptides in conjunction with biological assays. These studies provide new insights into the structure-activity relationships of β-defensins. Ion mobility-mass spectrometry (IM-MS) is used throughout this thesis to probe the tertiary structure of peptides in vacuo and, by inference, make conclusions about their conformations in solution prior to ionisation. Where appropriate, IM-MS is complemented by other techniques, including high performance liquid chromatography and circular dichroism spectroscopy. First, the importance of a C-terminal cysteine residue within the murine β-defensin Defb14 is investigated. The functional and structural implications of chemically modifying the cysteine residue are examined. Second, the N-terminal region of Defb14 is modified by the substitution and deletion of amino acids. Again, the effects on biological activity and structure are discussed. Finally, the functional and structural overlap of β-defensins with another family of proteins – the chemokines – is considered. The oligomerisation of β-defensins and their interaction with glycosaminoglycans is of particular interest: structural data for human β-defensins 2 and 3 in the absence and presence of polysaccharides are presented.

Defensins are small cysteine-rich, cationic peptides that play an essential role in the innate immune system of virtually all life forms, from insects and plants to amphibians and mammals. Defensins are mainly an innate immunity element, exhibiting antibacterial activities by disrupting the cell membrane of a wide range of organisms (Cole et al. 2002). Defensins also affect certain adaptive immune responses, including enhancing phagocytosis, promoting neutrophil recruitment, and enhancing the production of proinflammatory cytokines. The aim of this thesis is to make a comprehensive and accurate subfamily classification of the defensin gene family, primarily by using a library of Hidden Markov Models (HMMs). In this project the subfamily classification of the defensin gene family is primarily based on a constructed library of HMMs. Results: Sets of known defensins were organized in placed in 84 clusters using the clustering and alignment tool, FlowerPower. The clusters were further classified as mammalian alpha- or beta-defensins, plant defensin, insect defensin and defensin MGD. This classification was based on significant cluster hits against the Structural Classification of Proteins (SCOP) database and species distribution. Based on the relative positions of disulfide bonds and constructed Multiple Sequence Alignments (MSAs) some sequences were classified as belonging to the sperm– and theta-defensin subfamilies. Compared to PFAM’s classification of defensins, the subfamily classification presented here is more informative. The library of HMMs has been made public via a web server that was used to automatically score and analyze input sequences against the created database of HMMs. This database and web server are expected to be useful to researchers working on various aspects of defensin action.

Infectious agents such as bacteria or viruses can grow rapidly. If a microorganism invades a host, it must be recognized rapidly and destroyed before it overwhelms the immune system. Limiting infection to a minimum in the early stage is critical for the outcome and the recovery from infection. The innate immune system has evolved to recognize a few highly conserved, constitutive structures present only in microorganisms, such as bacterial lipopolysaccharide (LPS), called pathogen-associated molecular patterns (PAMP). Toll-like receptors are the host receptors that recognize PAMP, ultimately activating a variety of transcription factors to induce expression of a wide spectrum of immune related genes, e.g. defensins. Defensins are antimicrobial peptides that play an important role in innate defense against microorganisms in plants and animals. Beta-defensins are the largest family of antimicrobial peptides, which can directly kill microorganisms and have regulatory effects on the immune system. Thirteen beta-defensins have been identified; however, the regulation of these genes has not been well-investigated in the chicken. The objective of this research was to understand constitutive and inducible gene expression of beta-defensins in chicken white blood cells. Real-time RT-PCR was used to quantify gene expression level before and after LPS stimulation. Transcription factor binding sites in the genes were identified to understand the gene expression regulation. From the expression profile results, most chicken beta-defensins had induced gene expression by LPS stimulation in the early phase (0- to 3-hour) and reduced gene expression in the late phase (3- to 8-hour). As for the level of gene expression, the results show that the induced gene expression in the early phase corresponded to the higher levels of expression at 3-hours after LPS stimulation, and the reduced gene expression in the late phase corresponded to the lower levels of gene expression at 8-hours after LPS stimulation.

<p>Defensins are small cysteine-rich, cationic peptides that play an essential role in the innate immune system of virtually all life forms, from insects and plants to amphibians and mammals. Defensins are mainly an innate immunity element, exhibiting antibacterial activities by disrupting the cell membrane of a wide range of organisms (Cole et al. 2002). Defensins also affect certain adaptive immune responses, including enhancing phagocytosis, promoting neutrophil recruitment, and enhancing the production of proinflammatory cytokines.</p><p>The aim of this thesis is to make a comprehensive and accurate subfamily classification of the defensin gene family, primarily by using a library of Hidden Markov Models (HMMs). In this project the subfamily classification of the defensin gene family is primarily based on a constructed library of HMMs. Results: Sets of known defensins were organized in placed in 84 clusters using the clustering and alignment tool, FlowerPower. The clusters were further classified as mammalian alpha- or beta-defensins, plant defensin, insect defensin and defensin MGD. This classification was based on significant cluster hits against the Structural Classification of Proteins (SCOP) database and species distribution. Based on the relative positions of disulfide bonds and constructed Multiple Sequence Alignments (MSAs) some sequences were classified as belonging to the sperm– and theta-defensin subfamilies. Compared to PFAM’s classification of defensins, the subfamily classification presented here is more informative. The library of HMMs has been made public via a web server that was used to automatically score and analyze input sequences against the created database of HMMs. This database and web server are expected to be useful to researchers working on various aspects of defensin action.</p>

Porphyromonas gingivalis produces proteases capable of degrading cytokines, host heme proteins, and some antimicrobial peptides. In this work, I show that P. gingivalis culture supernatants fully or partially degrade human neutrophil peptide alpha-defensins and human beta-defensins after 30 minutes. This observation suggests that proteases from P. gingivalis degrade defensins and this activity could abrogate defensin-related innate immune functions.

Thesis (Master of Applied Sciences in Chemistry)--Cape Peninsula University of Technology, 2018. / Antimicrobial Peptides (AMPs) are short peptides of about 45 - 54 amino acids that exhibit antibacterial and antifungal activities. Plant defensin is a type of AMP in plants which belong to a family of cationic peptides with a characteristic 3D folding pattern held in place by four disulfide bridges. AMPs especially defensins have been identified to have a huge biotechnological potential and are being patented for many applications. The aim of this work was to clone an antimicrobial peptide from Vigna subterranea and characterise it with bioinformatics analysis. 4 sets of primers were synthesized according to the sequences of conserved regions in AMPs i.e. defensins from legumes like Vigna unguiculata, Vigna radiata, Cicer arietinum and Cajanus cajan, amongst others, which have defensins with only a few sequence differences. The primers were designated VsDef P1 to P4. Using Vigna subterranea total genomic DNA as a template, fragments of expected sizes were successfully amplified and cloned into the pDRIVE vector and used to transform Escherichia coli JM109 cells in each case. Representative clones were sequenced and analysed using BLAST from National Center for Biotechnology Information. However, only the VIG clone was shown to be a bona fide defensin (over 90% identity, E-value of 1ex102, 99% query coverage of the nucleotide sequence, compared to Vigna unguiculata defensin). Based on this high sequence identity, a new pair of primers VsDef P5 was designed based on the Vigna unguiculata defensin sequence to specifically amplify the complete Vigna subterranea defensin gene, hereafter called VsDef1. Attempts to clone VsDef1 were however unsuccessful, and evidence of clone deletion and insert re-arrangement of insert DNA was observed. Direct sequencing of the PCR product demonstrated that it was indeed the complete VsDef1 pre-protein, composed of 433 nucleotides. In silico translation and analysis showed that VsDef1 has an intron at position 105 − 259 of the nucleotide sequences and encodes for a 78 amino acid peptide. Phylogenetic analysis revealed to be similar to the sequence of the defensins for Vigna unguiculata (96%), Vigna radiata (95%), Vigna angularis (95%) and Phaseolus vulgaris (93%) on the NCBI database. The three - dimensional structure of the peptide was modelled with SWISS-MODEL expasy and the structure was found to include one α- and three β domains, similar to those of other defensins. The failure to identify VsDef1 clone in a V. subterranea library and the failure to recover its cDNA clone are consistent with the hypothesised toxicity of VsDef1 to Escherichia coli. It is suggested that a different host, such as yeast, should be used in the future. The VsDef1 mRNA levels in germinating V. subterranea seeds was however successfully investigated using real-time reverse transcription quantitative PCR. VsDef1 mRNA is present in both the testa and embryo of dry seed and will persist through the early stages of seedling growth. This demonstrates the importance of VsDef1 in fighting off infection during germination in order to ensure successful germination. It is therefore essential to characterise more antimicrobial peptides from V. subterranea. The diversity of AMPs and their patterns of expressed genes will enable understanding of complex regulatory networks, which will likely enable identifying of genes involved in diseases and new biological processes.

Bovine herpesvirus-1 (BoHV-1) causes recurrent respiratory and genital infections in cattle; and predisposes them to lethal secondary bacterial infections. Vaccination is a primary strategy to prevent and reduce the severity of disease associated with BoHV-1, and to reduce virus transmission. While modified live (MLV) or killed (KV) BoHV-1 vaccines exist, these are expensive to produce, can cause disease (MLV) or may be ineffective (KV). Development of a DNA vaccine for BoHV-1 has the potential to address these shortcomings, but the very small amount of antigen expressed after DNA immunization presents a barrier to successful immunization of large animals. Engineering the vaccine to target this limited quantity of antigen to dendritic cells (DCs), the cells that prime immune responses, by attracting immature DCs (iDCs) to the vaccination site, is one way that DNA vaccine efficacy might be improved. Beta (β)-defensins are chemotactic peptides that, in studies with mice, improve induction of immune responses to DNA vaccines and this is due, at least in part, to their ability to attract iDCs to the site of vaccination. Accordingly, the objective of the studies described in this thesis was to determine whether using a bovine β-defensin in a DNA vaccine would enhance immune responses to the vaccine and subsequently protect cattle upon challenge with BoHV-1. First I characterized the bovine iDC and then used these cells to screen a panel of synthesized bovine β-defensins for chemotactic activity. The results showed that bovine neutrophil β-defensin (BNBD) 3, BNBD9 and enteric β-defensin (EBD) were equally the most chemotactic of the fourteen synthesized peptides for bovine iDCs. Because BNBD3 is the most abundant of the thirteen BNBDs and was able to attract CD1+ DCs when injected into the skin, I chose BNBD3 as the peptide I would use for the rest of the project. Next I constructed plasmids that expressed BNBD3; either alone or as a fusion construct with the BoHV-1 antigen truncated glycoprotein D (tgD), and then tested the effects of the plasmids as vaccines in both mice and cattle. In cattle, the addition of BNBD3 as a fusion strengthened the Th1 bias and increased cell-mediated immune responses to the DNA vaccine but not antibody response or protection from BoHV-1 infection. Given that inefficient humoral immune responses have been implicated in a lack of protection from BoHV-1 challenge, these results suggested that the successful BoHV-1 DNA vaccine would need to induce a much stronger humoral response. Lastly I assessed the ability of BNBD3 to improve humoral responses to pMASIA-tgD when complexed with the DNA vaccine and found that the vaccine complexed at a nanomolar peptide to DNA ratio of 125:1 increased humoral responses of mice. In vitro, treatment of mouse bone-marrow DCs with BNBD3 induced phenotypic and functional maturation/activation. This is an important aspect for vaccination in the skin, since after uptake, the DC must “mature” in order to traffic from the site of vaccination to the draining lymph node where induction of antigen-specific responses, by activated DCs, takes place. The findings in this thesis show that bovine β-defensins are chemotactic for bovine iDCs. I also show that using a bovine β-defensin as a fusion construct in a DNA vaccine enhances cell mediated but not humoral responses of cattle and yet this vaccine is protective against BoHV-1 challenge. I demonstrate that a bovine β-defensin, when used as a peptide to complex an antigen-encoding plasmid, can increase humoral responses. My work shows a multifunctional ability of bovine β-defensins to modulate and increase immune responses and suggests that bovine β-defensins likely have further untapped potential to enhance efficacy of DNA vaccines for large animals.