Cationic antimicrobial peptides (CAMPs) are produced by plants, animals and bacteria to protect their host against antagonistic microbes. The antitheses of selective antibiotics, these peptides are drawn by electrostatic and hydrophobic interactions to targets as diverse as the bacterial membrane, nucleic acids and serum proteins. This lack of specificity is their greatest strength, as mutations to single genes rarely lead to bacterial resistance. Resistance may be conferred by large scale alterations in cell envelope composition, which generally reduces bacterial fitness in the absence of peptide. Clinical applications of natural CAMPs are limited, as the peptides are toxic to mammalian cells and rapidly inactivated in vivo by serum albumin and proteases. Faced with these challenges we have prepared a number of CAMP analogues, with the goal of creating lead compounds for further development of antibacterial therapeutics. Much of our work has focused on ultrashort lipopeptides and lipopeptoids, which have properties similar to natural CAMPs and extremely abbreviated sequences. The simple structure of these scaffolds allows rapid creation of CAMP analogues in a brief period of time, allowing us to rapidly explore the structural requirements for CAMP activity. The balance of this work focuses on imparting CAMP-like behaviour to known antibiotics, in order to expand their spectrum of susceptible bacteria and combat the development of drug-resistant bacteria. In particular, the aminoglycosides neomycin and tobramycin have been fused to phenolic disinfectants such as triclosan and biclotymol, in order to improve their diffusion across the bacterial envelope and activity against Gram-negative bacteria.

<p>Outer membrane vesicles are an important constitutive product of all Gram-negative bacteria. Bacteria have evolved many responses to alleviate all different types of stress. The primary objective of this dissertation is to investigate the role of outer membrane vesicles (OMVs) as a method by which Gram-negative bacteria can quickly act to protect themselves against particular threats. Generally, we find that stressors whose primary effect is on the outer membrane can be protected against by OMVs. Throughout this study, a variety of different microbiological and biochemical methods are used to answer key questions in the innate ability of OMVs to protect against particular antimicrobials. Using Escherichia coli as well as Pseudomonas aeruginosa as model organisms we tested the ability of purified vesicles from each species to protect themselves and other hosts. Using bacteriophage T4, we investigated the ability of OMVs purified from E. coli to adsorb phage as well as how this interaction affected the efficiency of infection. We found that OMVs are protective against antimicrobial peptides, as well as bacteriophage. In the course of understanding this protection we also observed and characterized the cross species effects of both OMV protection as well as phage infection. Where typically a phage infects a specific species, we found that T4 associated OMVs treating a non-native host P. aeruginosa resulted in the production of a novel prophage. Upon further examination, we determined that this induction was occurring via a novel pathway that we attempted to further characterize by performing a genetic screen to identify genes important to this induction. The work within this dissertation fully supports the hypothesis of a regulated response to outer membrane acting stimuli, resulting in the induction of vesiculation and the adsorption of stressor in the extra-cellular milieu. This model of protection agrees with the idea of a bacterial innate defense system, which acts in the short term before the adaptive response can fully occur, resulting in a bridge between the untreated to the treated and resistant culture.</p> / Dissertation

Cationic antimicrobial peptides (CAMPs) are produced by plants, animals and bacteria to protect their host against antagonistic microbes. The antitheses of selective antibiotics, these peptides are drawn by electrostatic and hydrophobic interactions to targets as diverse as the bacterial membrane, nucleic acids and serum proteins. This lack of specificity is their greatest strength, as mutations to single genes rarely lead to bacterial resistance. Resistance may be conferred by large scale alterations in cell envelope composition, which generally reduces bacterial fitness in the absence of peptide. Clinical applications of natural CAMPs are limited, as the peptides are toxic to mammalian cells and rapidly inactivated in vivo by serum albumin and proteases. Faced with these challenges we have prepared a number of CAMP analogues, with the goal of creating lead compounds for further development of antibacterial therapeutics. Much of our work has focused on ultrashort lipopeptides and lipopeptoids, which have properties similar to natural CAMPs and extremely abbreviated sequences. The simple structure of these scaffolds allows rapid creation of CAMP analogues in a brief period of time, allowing us to rapidly explore the structural requirements for CAMP activity. The balance of this work focuses on imparting CAMP-like behaviour to known antibiotics, in order to expand their spectrum of susceptible bacteria and combat the development of drug-resistant bacteria. In particular, the aminoglycosides neomycin and tobramycin have been fused to phenolic disinfectants such as triclosan and biclotymol, in order to improve their diffusion across the bacterial envelope and activity against Gram-negative bacteria.

Anurans are declining worldwide because of the spread of Batachochytrium dendrobatidis (Bd), the fungus that causes chytridiomycosis. However, some frogs are resistant to this disease, and understanding why may be critical to saving those that are susceptible. In Peru, Gastrotheca excubitor is resistant to chytridiomycosis while Gastrotheca nebulanastes is susceptible. Two anuran skin defenses, symbiotic bacteria and antimicrobial peptides (AMPs), have demonstrated the ability to inhibit Bd in vitro when isolated from certain frogs. We tested if these defenses can explain the difference in susceptibility between the two Gastrotheca species. The cutaneous bacteria and AMPs of both species were collected, tested for their abilities to inhibit the growth of Bd, and analyzed for their compositions. Results indicate that 34%of the strains of skin bacteria from G. excubitor were able to inhibit the growth of Bd whereas only 10% isolated from G. nebulanastes were effective. Gastrotheca excubitor also has stronger anti-Bd skin bacteria. Neither frog species has peptide mixtures capable of completely inhibiting Bd, and overall species did not differ in the anti-Bd abilities of their peptides. These results suggest that the chytridiomycosis resistance experienced by G. excubitor may be attributed to its skin bacteria.

Modern medicine is challenged continuously by the increasing prevalence of multi-drug resistant bacteria. Therefore, the development of alternatives to traditional antibiotics is an urgent necessity. Cationic antimicrobial peptides (CAMPs) are components of the innate immune defense system. Histones, generally known as proteins that package and regulate the transcription of DNA, share all of the essential antimicrobial traits of CAMPs, and could be promising alternatives to antibiotics. In this study, I investigated the antimicrobial properties of nucleated-erythrocyte-specific linker histone H5 and its derived peptides. Histone H5 was extracted and purified from chicken erythrocytes using an acid extraction followed by ion exchange chromatography using a step salt gradient; the purity (>95%) was verified by densitometry and proteomics analysis. Purified histone H5 demonstrated potent antimicrobial activity against various Gram-positive and Gram-negative planktonic bacteria, including resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), as well as anti-biofilm activity against Listeria monocytogenes and Pseudomonas aeruginosa. Furthermore, scanning electron microscopy (SEM) revealed significant damage to L. monocytogenes and P. aeruginosa bacterial cell surfaces after histone H5 treatment. The potential for histone toxicity towards mammalian cells was investigated with a hemolytic assay which determined that even at the highest concentration tested (1 mg/mL), histone H5 was non-hemolytic. An in silico analysis determined the predicted antimicrobial domain of histone H5 of which six histone H5-derived peptides with potential antimicrobial activity were identified. These six histone H5-derived peptides were synthesized and tested against bacterial pathogens to determine their antimicrobial properties. Although the H5-derived peptides were identified within the predicted antimicrobial domain of histone H5, they did not possess more potent antimicrobial activity than the full length protein. Overall, this study demonstrates that histone H5 and histone H5-derived peptides could be promising candidates in the development of novel anti-infective therapeutics.

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

Amphibian metamorphosis is facilitated solely by thyroid hormones (THs), L-thyroxine (T4) and 3,5,3’-triiodothyronine (T3). TH modulates the remodeling of many different organs and systems in the body of developing tadpoles, including the immune system. Previous research found evidence of T4 action on direct-response genes in outer ring deiodinase-poor premetamorphic tadpole tail fin and liver without the required conversion to T3 described by current TH dogma. The mechanisms of environmental endocrine disrupting chemicals (EDCs) may be better understood by expanding our understanding of the transcriptomic effects of both forms of THs and how they relate to estrogen signaling. Furthermore, analysis of TH-modulation of the immune system may enable a greater understanding of the devastating effects of amphibian pathogens such as Ranavirus. Premetamorphic Rana (Lithobates) catesbeiana tadpoles were exposed to physiological concentrations of T4, T3, or 17-beta-estradiol (E2) through water bath immersion. qPCR analysis was performed to assess the response of canonical TH-responsive genes thra, thrb, and thibz to these hormones in the liver and tail fin tissues of bullfrog tadpoles. E2 treatment did not elicit a response in these gene transcripts in either tissue. T3 treatment in the tail fin elicited an overall stronger response than T4, while T4 treatment in the liver recapitulated results consistent with non-genomic mechanisms of T4 signaling for thrb and thibz transcripts. Illumina Hiseq2500 was used to sequence RNA isolated from hormone-treated premetamorphic tadpole liver and tail fin tissues to assess differential transcriptomic responses and identify TH-responsive immune system-associated transcripts. The impact of TH-treatment on the general immune system in the liver and tail fin transcriptomes was also analyzed using RNA-seq data. We found that E2 modulates at least some shared TH pathways in the liver, but none in the tail fin and that the tail fin transcriptome is more affected by T3, while the liver transcriptome is more affected by T4. Additionally, evidence of immune system modulation by both THs was found in both the liver and tail fin transcriptomes. Antimicrobial peptides (AMPs) are an important component of the amphibian immune response. Details regarding the regulation, synthesis, and expression of AMPs remain obscure, although evidence of TH-modulation of specific AMPs has been identified, as well as evidence of increased expression of AMPs throughout metamorphosis. Frog skin is a prolific source of AMPs that may prove useful in the quest for alternative antimicrobial agents in the face of antibiotic resistance. Identification of new AMPs is hindered by the practical limitations of classical protein-based discovery approaches. By using known AMP characteristics and common ¬AMP properties, we developed a high throughput bioinformatics approach predicated on the use of R. catesbeiana genome resources. We mined these resources and identified novel and known AMPs that exhibited verified antimicrobial activity against various bacterial organisms. This thesis sought to elucidate the differential and modulatory effects of both forms of TH on a transcriptomic level and in the context of immunity, and to examine the utility of the bullfrog transcriptome and genomics resources in identifying and characterizing novel bullfrog-derived AMPs and elucidating aspects of AMP expression. / Graduate / 2018-12-08

学位プログラム名: 京都大学大学院思修館 / 京都大学 / 0048 / 新制・課程博士 / 博士(総合学術) / 甲第21231号 / 総総博第3号 / 新制||総総||1(附属図書館) / 京都大学大学院総合生存学館総合生存学専攻 / (主査)教授 竹本 佳司, 特定教授 大嶌 幸一郎, 教授 大野 浩章 / 学位規則第4条第1項該当 / Doctor of Philosophy / Kyoto University / DFAM

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