The emergence of pathogens resistant to available drug therapies is a pressing global health problem. Antimicrobial peptides (AMPs) may potentially form new therapeutics to counter these pathogens. AMPs are key components in the mammalian innate immune system and are responsible for both direct killing and immunomodulatory effects in host defense against pathogenic organisms. This thesis describes computational methods for the identification of novel natural and synthetic AMPs. A bioinformatic resource was constructed for classification and discovery of gene- coded AMPs, consisting of a database of clustered known AMPs and a set of hidden Markov models (HMMs). One set of 146 clusters was based on the mature peptide sequence, and one set of 40 clusters was based on propeptide sequence. The bovine genome was analyzed using the AMPer resources, and 27 of the 34 known bovine AMPs were identified with high confidence and up to 69 AMPs were predicted to be novel peptides. One novel cathelicidin AMP was experimentally verified as up-regulated in response to infection in bovine intestinal tissue. A chemoinformatic analysis was performed to model the antibacterial activity of short synthetic peptides. Using high-throughput screening data for the activities of over 1400 peptides of diverse sequence, quantitative structure-activity relation (QSAR) models were created using artificial neural networks and physical characteristics of the peptide that included three-dimensional atomic structure. The models were used to predict the activity of a set of approximately 100,000 peptide sequence variants. After ranking the predicted activity, the models were shown to be very accurate. When 200 peptides were synthesized and screened using four levels of expected activity, 94% of the top 50 peptides expected to have the highest level of activity were found to be highly active. Several promising candidates were synthesized with high quality and tested against several multi- antibiotic-resistant pathogens including clinical strains of Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. These peptides were found to be highly active against these pathogens as determined by minimal inhibitory concentration; this serves as independent confirmation of the effectiveness of high-throughput screening and in silico analysis for identifying peptide antibiotic drug leads.

A new paradigm for the treatment of infectious disease is through the modulation of innate immune responses. In this capacity, host defense peptides (HDPs) and synthetic Toll-like receptor 9 (TLR9) ligands have the greatest demonstrated potentials. The work presented here considers mechanisms for the improvement of these treatments through optimization, or in the case of HDPs the minimization, of the interactions of these ligands with sensory receptors.<p>Toll-like Receptor 9 activates the innate immune system in response to microbial DNA or immune-modulating oligodeoxynucleotides. While cell stimulation experiments demonstrate the preferential activating ability of CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions regarding the sequence-specificity of TLR9 ligand binding. To address this discrepancy the characterization of human TLR9 ligand binding properties is reported. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity of the receptor is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. <p>Host defense peptides are among the leading candidates to combat antibiotic resistant bacterial strains. Recently, HDPs have been demonstrated to function as ligands for the bacterial sensory kinase PhoQ resulting in the induction of virulence and adaptive responses. Thus, concerns have been raised regarding therapeutic applications of HDPs. Here a methodology is described that permits discrimination and quantification of the distinct, but related, peptide behaviors of direct antimicrobial activity and PhoQ ligand potential. Utilizing peptide derivatives of the model HDP Bac2A it is demonstrated that antimicrobial efficiency is significantly, and inversely, related to PhoQ ligand efficacy. This provides a rational basis for HDP selection with greater therapeutic potential and minimized potential for initiation of bacterial resistance.

The emergence of pathogens resistant to available drug therapies is a pressing global health problem. Antimicrobial peptides (AMPs) may potentially form new therapeutics to counter these pathogens. AMPs are key components in the mammalian innate immune system and are responsible for both direct killing and immunomodulatory effects in host defense against pathogenic organisms. This thesis describes computational methods for the identification of novel natural and synthetic AMPs. A bioinformatic resource was constructed for classification and discovery of gene- coded AMPs, consisting of a database of clustered known AMPs and a set of hidden Markov models (HMMs). One set of 146 clusters was based on the mature peptide sequence, and one set of 40 clusters was based on propeptide sequence. The bovine genome was analyzed using the AMPer resources, and 27 of the 34 known bovine AMPs were identified with high confidence and up to 69 AMPs were predicted to be novel peptides. One novel cathelicidin AMP was experimentally verified as up-regulated in response to infection in bovine intestinal tissue. A chemoinformatic analysis was performed to model the antibacterial activity of short synthetic peptides. Using high-throughput screening data for the activities of over 1400 peptides of diverse sequence, quantitative structure-activity relation (QSAR) models were created using artificial neural networks and physical characteristics of the peptide that included three-dimensional atomic structure. The models were used to predict the activity of a set of approximately 100,000 peptide sequence variants. After ranking the predicted activity, the models were shown to be very accurate. When 200 peptides were synthesized and screened using four levels of expected activity, 94% of the top 50 peptides expected to have the highest level of activity were found to be highly active. Several promising candidates were synthesized with high quality and tested against several multi- antibiotic-resistant pathogens including clinical strains of Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. These peptides were found to be highly active against these pathogens as determined by minimal inhibitory concentration; this serves as independent confirmation of the effectiveness of high-throughput screening and in silico analysis for identifying peptide antibiotic drug leads.

mPIP is a mouse homologue of human PIP/GCDFP-15 which is an established marker of both malignant and benign pathological conditions of the mammary gland. mPIP gene expression has been identified in both lacrimal and salivary glands of healthy mice and the mPIP protein has been detected in saliva. The mPIP protein has been found to bind oral bacteria, showing the highest affinity for streptococci, suggesting a potential function of mPIP in the non-immune host defense in the mouse oral cavity. Since the exact functions of mPIP are still unknown, we examined the roles of mPIP through both in vitro and in vivo studies, specifically to address the possible role of this protein in non-immune host response through modulating the oral flora. The in vitro studies were primarily focused on elucidation of the consequences of interaction between mPIP and oral bacteria, in particular to examine whether mPIP plays a role in bacterial aggregation. The in vivo studies addressed the roles of mPIP through the analysis of an mPIP knockout mouse model generated in our laboratory. Following confirmation of the null mutation, the delineating the phenotype of this model was pursued through morphopathological analysis as well as examination of the impact of the lack of mPIP on the mouse oral flora. The null mutation in the mPIP knockout mice was confirmed by both the gene and protein analysis. Histological analysis revealed lymphocytic proliferation in both the submaxillary and prostate glands of the mPIP knockout mice. In addition, both quantitative and composition differences in the oral flora of mPIP knockout mice were identified when compared with wild-type controls. Specifically, a higher proportion of the oral bacteria of mPIP knockout mice were found to belong to genus Streptococcus and certain genera were found to be absent from the oral cavity of these mice. The effect of knockout mouse saliva, which lacks mPIP, on the aggregation of oral bacteria was compared to wild-type mouse saliva. Our data suggests that mPIP contributes to saliva-induced bacterial aggregation. While oral flora has multiple functions, including protection against infection, mPIP might play a role in the non-innate host defense through modulating the resident oral flora in the mouse. The identification of lymphocytic proliferation in submaxillary and prostate glands of mPIP knockout mice suggests that mPIP might also interfere with lymphocyte activity, playing a possible immunomodulatory role.

mPIP is a mouse homologue of human PIP/GCDFP-15 which is an established marker of both malignant and benign pathological conditions of the mammary gland. mPIP gene expression has been identified in both lacrimal and salivary glands of healthy mice and the mPIP protein has been detected in saliva. The mPIP protein has been found to bind oral bacteria, showing the highest affinity for streptococci, suggesting a potential function of mPIP in the non-immune host defense in the mouse oral cavity. Since the exact functions of mPIP are still unknown, we examined the roles of mPIP through both in vitro and in vivo studies, specifically to address the possible role of this protein in non-immune host response through modulating the oral flora. The in vitro studies were primarily focused on elucidation of the consequences of interaction between mPIP and oral bacteria, in particular to examine whether mPIP plays a role in bacterial aggregation. The in vivo studies addressed the roles of mPIP through the analysis of an mPIP knockout mouse model generated in our laboratory. Following confirmation of the null mutation, the delineating the phenotype of this model was pursued through morphopathological analysis as well as examination of the impact of the lack of mPIP on the mouse oral flora. The null mutation in the mPIP knockout mice was confirmed by both the gene and protein analysis. Histological analysis revealed lymphocytic proliferation in both the submaxillary and prostate glands of the mPIP knockout mice. In addition, both quantitative and composition differences in the oral flora of mPIP knockout mice were identified when compared with wild-type controls. Specifically, a higher proportion of the oral bacteria of mPIP knockout mice were found to belong to genus Streptococcus and certain genera were found to be absent from the oral cavity of these mice. The effect of knockout mouse saliva, which lacks mPIP, on the aggregation of oral bacteria was compared to wild-type mouse saliva. Our data suggests that mPIP contributes to saliva-induced bacterial aggregation. While oral flora has multiple functions, including protection against infection, mPIP might play a role in the non-innate host defense through modulating the resident oral flora in the mouse. The identification of lymphocytic proliferation in submaxillary and prostate glands of mPIP knockout mice suggests that mPIP might also interfere with lymphocyte activity, playing a possible immunomodulatory role.

The emergence of pathogens resistant to available drug therapies is a pressing global health problem. Antimicrobial peptides (AMPs) may potentially form new therapeutics to counter these pathogens. AMPs are key components in the mammalian innate immune system and are responsible for both direct killing and immunomodulatory effects in host defense against pathogenic organisms. This thesis describes computational methods for the identification of novel natural and synthetic AMPs. A bioinformatic resource was constructed for classification and discovery of gene- coded AMPs, consisting of a database of clustered known AMPs and a set of hidden Markov models (HMMs). One set of 146 clusters was based on the mature peptide sequence, and one set of 40 clusters was based on propeptide sequence. The bovine genome was analyzed using the AMPer resources, and 27 of the 34 known bovine AMPs were identified with high confidence and up to 69 AMPs were predicted to be novel peptides. One novel cathelicidin AMP was experimentally verified as up-regulated in response to infection in bovine intestinal tissue. A chemoinformatic analysis was performed to model the antibacterial activity of short synthetic peptides. Using high-throughput screening data for the activities of over 1400 peptides of diverse sequence, quantitative structure-activity relation (QSAR) models were created using artificial neural networks and physical characteristics of the peptide that included three-dimensional atomic structure. The models were used to predict the activity of a set of approximately 100,000 peptide sequence variants. After ranking the predicted activity, the models were shown to be very accurate. When 200 peptides were synthesized and screened using four levels of expected activity, 94% of the top 50 peptides expected to have the highest level of activity were found to be highly active. Several promising candidates were synthesized with high quality and tested against several multi- antibiotic-resistant pathogens including clinical strains of Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. These peptides were found to be highly active against these pathogens as determined by minimal inhibitory concentration; this serves as independent confirmation of the effectiveness of high-throughput screening and in silico analysis for identifying peptide antibiotic drug leads. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

No description available.

Bacterial infections have posed a serious threat to the public health due to the significant rise of the infections caused by antibiotic-resistant bacteria. There has been considerable interest in the development of antimicrobial agents which mimic the natural HDPs, and among them biodegradable polymers are newly discovered drug candidates with ease of synthesis and low manufacture cost compared to synthetic host defense peptides. Herein, we present the synthesis of biocompatible and biodegradable polymers including polycarbonate polymers, unimolecular micelle hyperbranched polymers and dendrimers that mimic the antibacterial mechanism of HDPs by compromising bacterial cell membranes. The developed amphiphilic polycarbonates are highly selective to Gram-positive bacteria, including multidrug-resistant pathogens and the unimolecular micelle hyperbranched polymers showed promising broad-spectrum activity. However, lipidated amphiphilic dendrimers with low molecular weight display potent and selective antimicrobial activity against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains. In addition to antibacterial activity against planktonic bacteria, these dendrimers were also shown to inhibit bacterial biofilms effectively. These class of polymers may lead to a useful generation of antibiotic agents with practical applications.

The lactoperoxidase (LPO) antimicrobial system has been shown to play an important role in maintaining sterile conditions in several tissues including the mammary gland, the salivary gland, and the airway. The LPO system in the airway consists of the enzyme LPO and its substrates hydrogen peroxide and an anion. LPO catalyzes the oxidation of a halide or pseudohalide ion for example SCN-or I- by hydrogen peroxide producing a product, OSCN- or OI- which have antibacterial, antifungal, and antiviral properties. In order to have a functional antimicrobial system all the components need to be present at appropriate concentrations. The LPO system has been suggested to be deficient in cystic fibrosis. There are three possible regulatory mechanism of this antimicrobial system and these involve the secretion and availability of the three components of the LPO system in the luminal fluid. The studies presented in this dissertation examine two of the possible regulatory mechanisms of the LPO system in the airway; the availability and transport of SCN- to the luminal surface, and the expression of LPO. The knowledge obtained from these studies could be utilized to develop treatments to control infection in diseases characterized by chronic infections such as cystic fibrosis.

Dendritic cells (DCs) are potent antigen presenting cells (APCs) that link the innate and adaptive immune system by their unique ability to induce and direct immune responses towards various T helper (Th)-type of immune responses such as Th1-, Th2-, Th9-, Th17-, Th22- or T regulatory (TR). The type of Th response generated very much depends on the nature of the antigen encountered and allows for an effective and proficient immune response. For example, Th1 responses are used to clear intracellular pathogens while Th2 responses are needed to clear extracellular pathogens The ability to specifically modulate Th-responses is an area of intense research, as it allows for the development of more effective vaccines and immunotherapeutics. Immunomodulation of DCs is one strategy by which specific Th-type immune responses may be tailored. Current research is focused on identifying agents that have the capacity to immunomodulate DCs such as host defense peptides (HDPs). Apart from their anti-microbial activities, HDPs have a number of immune functions including recruitment and subsequent activation of DCs. The goal of this study was to examine the immunomodulatory effects of HDPs on porcine DC functions. This research was part of a larger multinational research project to develop a novel adjuvant platform for single-immunization vaccines against pertussis in neonates. The pig model was used for this research because of its physiological similarities to humans and the recently developed pertussis infection model in young piglets. A series of experiments was conducted to characterize and describe porcine DC functions. Two subsets of DCs were successfully characterized and tested for their response to stimulation with HDPs. Initial results demonstrated a minimal effect of HDPs on DC functions, therefore we expanded the number of HDPs used to include both synthetic derivatives of HDPs known as innate defense regulators (IDRs) and naturally- occurring HDPs. We examined these effects on peripheral blood mononuclear cells (PBMC) in vitro and found that HDPs induce expression of the chemokine interleukin (IL)-8, which resulted in PBMC recruitment in vitro. We then proceeded to evaluate the HDPs in vivo by intradermally administering them into the flank of pigs. Surprisingly, treatment with the HDPs did not result in recruitment of neutrophils in vivo. We also examined the effects of formulating IDR-1002 as an adjuvant with the academic antigen Keyhole Limpet Hemocyanin (KLH) on the development of KLH-specific immune responses in vaccinated pigs. While there was no difference in antibody titers between vaccinated and control animals, we found that co-formulation with IDR-1002 decreased both antigen-specific and mitogen-induced proliferation in KLH/IDR-1002 vaccinated animals as long as four weeks post-treatment. These results demonstrate that specific IDRs can suppress certain aspects of the pro-inflammatory immune response making them potentially highly versatile tools to modulate and tailor the immune response in disease states characterized by a pro-inflammatory component.