Dissertations / Theses on the topic 'Antimicrobial PEPTIDES IN VIVO'

Despite the success of modern medicine in treating infections, infectious diseases remain a major source of morbidity and mortality worldwide. The evolution of antibiotic resistant strains of bacteria means that new innovations in therapeutics must be pursued to combat this emerging threat. A novel approach is to utilize the anti-infective properties of endogenous host defense peptides by creating smaller synthetic peptides with enhanced protective activities. Some of these peptides directly kill bacteria and many display varied immunomodulatory activities, enhancing the host innate immune response to more effectively clear an infection. Here I examined the efficacy of several synthetic peptides in a murine model of invasive bacterial infection, induced by the Gram positive bacterium Staphylococcus aureus. Several peptides were able to significantly reduce peritoneal bacterial load in vivo by up to 4-logs relative to the controls, either through direct antibacterial killing or immunomodulatory activity. The latter class was studied in more detail; in particular, the peptides IDR-1 and 1002 displayed significant immunomodulatory effects in vivo. Both peptides were able to significantly induce the proinflammatory chemokines MCP-1, RANTES and KC, as well as increased recruitment of neutrophils and monocytes to the site of infection. These effects were not dependent on live bacteria, as heat inactivated S. aureus was also able to induce chemokines and cell migration. Mice that had been depleted of macrophages did not respond to peptide treatment, indicating that macrophages are an important effector cells through which immunomodulatory peptides counter infections. These results suggest that synthetic peptides have the potential to become a viable treatment option for bacterial infections.

2009/2010
The antimicrobial peptides (AMPs) are an important component of the innate defense against invading microorganisms, are widespread in nature and may have multiple and diversified mechanisms of bactericidal action. In addition to their direct antimicrobial activity the are also involved in other biological processes. The aim of this project was to investigate the in vivo activity of Bac7(1-35), a bovine proline-rich antimicrobial peptide, having in mind its possible use as a lead compound for the development of novel anti-infective agents. Before moving to animal models of infection, the in vitro stability of the peptide in the presence of murine and human serum or plasma as well as its biodistribution in mouse were investigated. Antibacterial activity assays against Salmonella enterica showed that the presence of murine blood components largely inhibits the antibacterial activity of the peptide. On the contrary, in human serum and plasma Bac7(1-35) maintains its efficacy. This is due to the more rapid degradation by proteases of murine blood. The in vivo biodistribution of Bac7(1-35) was investigated by using a time-domain optical imaging apparatus and a fluorescently-labeled Bac7(1-35) derivative. The compound reaches the kidney and the bladder respectively 1 and 3 hours after i.p. injection. The in vivo and ex vivo analyses performed after 24 h confirm that the compound has been totally excreted. A mouse model of S. typhimurium infection was set up and used to test the therapeutic efficacy of Bac7(1-35). Treatment of infected mice with the peptide injected i.p. immediately after a lethal, intraperithoneal bacterial challenge, increased the mean survival time and reduced significantly the number of viable bacterial cells in liver and spleen of treated mice at 3 days post-inoculum. In 1/3 of the organ homogenates, the bacterial presence was undetectable and this result matches the percentage of cured animals (35%). In an attempt to improve its pharmacokinetic profile, the peptide was conjugated with polyethylene glycol (PEG), a non-toxic, non-immunogenic and FDA-approved polymer. Different strategies of pegylation have been considered to find the best method in terms of chemical yield and of maintenance of biological activity. Pegylation via a thioether ligation resulted the best strategy to obtain a slow active peptide release in human blood components with a reduced renal clearance and an increased bioavailability of Bac7(1-35), as biodistribution analyses demonstrated. Several important pathogens, such as S. enterica, cause disease by surviving and replicating within host cells. Since many AMPs have also immunomodulatory activities, we investigated the effect of Bac7(1-35) on the interaction between macrophages and Salmonella. We carried out phagocytosis assays with macrophages and the results suggest that Bac7(1-35) plays a positive modulatory effect on this function. Phagocytosis assays were also performed to determine if Bac7(1-35) could inhibit survival and replication of intracellular Salmonella. The results show that the peptide inhibits the replication of intracellular Salmonella, suggesting that it can exert its antibacterial activity within eukaryotic cells. Further studies are required to fully understand the details of the Bac7(1-35) biological activities. The results obtained provide encouraging evidence for future investigations on Bac7(1-35) and on the pegylated form Bac7(1-35)CAM-PEG20k also in other models of infection and with different intracellular pathogens.
XXIII Ciclo
1981

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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
As infecções relacionadas à assistência à saúde (IrAS), são uma das principais causas de mortalidade e aumento dos custos hospitalares em países desenvolvidos e em desenvolvimento. Nos casos em que um paciente adquire uma IrA e esta não é tratada adequadamente, a mesma pode evoluir para um quadro mais grave, podendo levar a sepse e consequentemente na maioria dos casos a morte. A sepse representa um importante problema de saúde pública, entretanto, um tratamento eficaz para esta síndrome ainda não foi encontrado. Peptídeos antimicrobianos foram relatados para modular a resposta à infecção bacteriana na sepse, independente dos mecanismos de resistência conhecidos para os antibióticos. Desta forma, procurou-se investigar a atividade imunomodulatória de duas formas de clavaninas sobre monócitos RAW 264.7, bem como a atividade antimicrobiana e a citotoxicidade in vitro. Em ensaios in vivo a genotoxicidade, a ação das clavaninas sobre a migração de neutrófilos e a eficácia do tratamento com as clavaninas em um modelo de infecção de ferida operatória por S. aureus e sepse polimicrobiana grave também foram avaliadas. Os estudos in vitro demostraram que as clavaninas inibiram completamente o crescimento de E. coli, K. pneumoniae e S. aureus, preveniram a secreção de citocinas pró-inflamatórias (TNF-α, IL-12) e NO, e aumentaram a secreção de IL-10. Além disso, as clavaninas não apresentaram atividade citotóxica sobre as células RAW 264.7. Nos experimentos in vivo, as clavaninas não apresentaram genotoxicidade, além de apresentarem-se quimoatraentes para neutrófilos. As clavaninas, também, reduziram significativamente as unidades formadoras de colônias de S. aureus no modelo experimental de ferida operatória, e reduziram a mortalidade dos animais sépticos em mais de 50%, quando comparados com animais controle. Devido à sua ação direta sobre células do sistema imune e microorganismos, as clavaninas aparentam ser compostos potenciais para o tratamento de infecções bacterianas graves como a sepse, demonstrando alto valor biotecnológico.
Healthcare-associated infections (HAIs) are a major cause of mortality, also increasing hospital costs in developed and developing countries. When a patient acquires HAIs and this is not properly handled, disease may clearly worst, leading to sepsis and consequently in major to death. Despite of sepsis represents an important public health problem, any effective treatment for this syndrome was obtained until now. In this view, antimicrobial peptides have been reported as modulators of immune response to bacterial infection in sepsis, with independent activity of mechanisms that lead to antibiotic. Thus, the immunomodulatory activity of two different forms of clavanins over RAW 264.7 monocytes, as well the in vitro antimicrobial and cytotoxic activities were here investigated. Furthermore, in vivo genotoxicity assays, the evaluation of clavanins activity on neutrophil migration and also the efficacy of treatment with clavanins in a wound S. aureus infection model and severe polymicrobial sepsis were also evaluated. Moreover, in vitro studies demonstrated that clavanins are able of inhibit the growth of E. coli, K. pneumoniae and S. aureus. Clavanins also prevented the secretion of proinflammatory cytokines (TNF-α, IL-12) and NO, and increased the IL-10secretion. In addition, clavanins showed none cytotoxicity on RAW 264.7 cells. During in vivo experiments, the clavanins showed no genotoxicity, showing however, a clear chemotactic effect for neutrophils. Clavanins also significantly reduced the colony-forming units of S. aureus in an experimental model of surgical wound infection and reduced the mortality of septic animals in more than 50 %, when compared to control group. Due to their direct activities over immune cells and microorganisms, clavanins are potential compounds for the treatment of serious bacterial infections such as sepsis, showing an enormous and remarkable biotechnological value.

In der humanen Haut spielen antimikrobielle Peptide (AP) bei Entzündungsgeschehen bakteriellen und nicht-bakteriellen Ursprungs eine bedeutende Rolle. Neben einer konstitutiven Expression AP können Zytokine deren vermehrte oder abgeschwächte Expression bewirken. In dieser Arbeit wurden die AP humanes β-Defensin (HBD) -2, HBD-3 und Psoriasin (PSO) in Bezug auf deren Expression in gesunder Haut und deren Modulation durch Zytokine in vivo anhand des xenogenen NOD-SCID-Maus-Transplantationsmodells untersucht. Nach erfolgreicher Transplantation von humaner Haut auf NOD-SCID Mäuse wurden die Zytokine TNF-α, IFN-γ und IL-13 in unterschiedlicher Dosierung einzeln und in Kombination intradermal appliziert. Für TNF-α konnte eine erhöhte Expression von HBD-2, HBD-3 und PSO auf RNA-Ebene mittels in-situ-Hybridisierung und Protein-Ebene mittels immunhistochemischer Nachweismethoden gezeigt werden. Eine erhöhte Expression nach Injektion von IFN-γ ließ sich für HBD-3 auf RNA-Ebene und Protein-Ebene und für HBD-2 auf RNA-Ebene erfolgreich belegen. PSO zeigte auf Protein-Ebene nach Modulation mit IFN-γ eine bei höherer Dosierung leicht abnehmende Expression. Eine Änderung der Expression durch IL-13 ließ sich nicht eindeutig belegen. In dieser Arbeit konnte die in der Literatur in vitro beschriebene Modulationsfähigkeit der untersuchten AP durch die verwendeten Zytokine in vivo belegt werden.

The goal of this project was to clone and express the antimicrobial peptide protegrin 1 (PG-1). Initially a yeast system was chosen but was discarded due to technical difficulties. Invitrogen's bacterial T7 expression system was chosen next to express the peptide. PG-1 expression was verified by anti-his immunoblot and then the peptide was purified by IMAC. Its activity was verified using a Bacillus subtillis radial diffusion assay.

Neste estudo foram realizados inicialmente, experimentos para avaliar a ação sinérgica do peptídeo antimicrobiano P34 com sobrenadantes de culturas de algumas bactérias lácticas selecionadas e isoladas de queijo Minas Frescal. Foi investigada a influência deste peptídeo na expressão de genes em L. monocytogenes e L. seeligeri, sua citotoxicidade em diferentes células eucarióticas e toxicidade “in vivo”. Também foram realizados alguns testes para avaliar a citotoxicidade do peptídeo antimicrobiano P40. A adição do peptídeo P34 no queijo provocou uma diminuição de até 3 ciclos logarítmicos na contagem de células viáveis de L. monocytogenes inoculada artificialmente. Um aumento significativo na expressão dos genes dltA, Imo 1695 e mptA de L. monocytogenes foi observado após 96 h com a presença do peptídeo P34 no queijo. A influência do peptídeo P34 na expressão de genes associados aos componentes do envelope celular de L. monocytogenes e L. seeligeri, promoveu um aumento não significativo nos níveis de transcrição de genes dltA, Imo1695 e mptA observados em L. monocytogenes após inoculação em placas e incubação por 24 h a 37°C ou 240 h a 4°C. Em L. seeligeri uma diminuição significativa na expressão do gene dltA foi observada. Os genes Imo1695 e mptA demonstraram uma diminuição significativa de sua expressão (2000 e 31872 vezes, respectivamente) na presença do peptídeo P34 e incubação por 24 h a 37°C. A inoculação da placa com o peptídeo P34 e incubação por 240 h a 4ºC não promoveu diminuição significativa da expressão do gene mptA. A citotoxicidade dos peptídeos P34 e P40 foi avaliada em células VERO, tratadas com diferentes concentrações (0,02 - 2,5 μg ml-1). Nos ensaios de MTT, NRU e LDH as EC50 para o peptídeo P34 foram 0.60, 1.25, 0.65 μg ml-1 e do peptídeo P40 foram 0,30, 0,51 e 0,57 μg ml-1, respectivamente. A atividade hemolítica em eritrócitos humanos foi de (5,8%) e (19%), respectivamente. Os efeitos sobre a viabilidade, motilidade e exocitose acrossomal de espermatozóides humanos também foram avaliadas para o peptídeo P34. Não houve reações de hipersensibilidade ou aumento significativo de títulos de anticorpos durante os experimentos imunogenicidade ou morte dos animais durante experimentos de toxicidade aguda ou subcrônica. A DL50 foi superior a 332,3 ± 0,76 mg/kg. Não foram observadas alterações significativas nos parâmetros bioquímicos séricos nos animais tratados com o peptídeo P34. Não foram detectados sinais de possível toxicidade nos animais do grupo tratado com 0,825 mg/ kg/dia do peptídeo P34. Neste grupo apenas alterações histológicas no baço com a presença de megacariócitos foram observadas. A partir destes resultados evidencia-se o potencial do peptídeo P34 para ser utilizado como bioconservante em alimentos.
In this study initial experiments were performed to evaluate synergistic action of the antimicrobial peptide P34 and culture supernatants of some selected lactic acid bacteria isolated from Minas Frescal cheese. The influence of this peptide in the expression of genes in L. monocytogenes and L. seeligeri, their cytotoxicity in differents eukaryotic cells and “in vivo” toxicity was investigated. Also, some tests were carried out o evaluate the cytotoxicity of the antimicrobial peptide P40. The peptide P34 caused a decrease of up to 3 log cycles in viable counts of L. monocytogenes artificially inoculated in cheese. A significant increase in expression of genes dltA, Imo1695 mptA of L. monocytogenes was observed after 96 h incubation of the peptide P34 in cheese. The influence of peptide P34 on the expression of genes associated to components of cell envelope of L. monocytogenes and L. seeligeri, promoted a non significant increase in the levels of transcription of genes dltA, Imo1695 and mptA were observed after incubation of L. monocytogenes for 24 hs at 37°C and 240 hs at 4°C in plates. In L. seeligeri a significant decrease was observed in gene expression dltA. The gene Imo1695 showed a significant decrease in its expression (2000-fold) after inoculation with the peptide P34. A significant decrease of expression was also observed for the gene mptA (31872 - times) after inoculation with the peptide P34 and incubation for 24 hours at 37°C. The inoculation of the plate with the P34 peptide and incubated for 240 hrs at 4°C, showed a non-significant decrease of gene expression. The cytotoxicity of the peptide P34 and P40 was assessed in VERO cells treated with different concentrations (0.02 - 2.5 μg ml- 1). In MTT, NRU and LDH assays the EC50 to the peptide P34 were 0.60, 1.25, 0.65 μg ml-1 and the peptide P40 were 0.30, 0.51 and 0.57 μg ml-1, respectively. The hemolytical activity on human erythrocytes was of (5.8%) and (19%), respectively. The effects on viability, motility and acrosomal exocytosis of humam sperm were also evaluated for peptideP34. There were no hypersensitivity reactions or significant increase in antibody titer during the immunogenicity experiment or death of animals during the acute or subchronic toxicity tests. The LD50 was more the 332.3 ± 0.76 mg/kg. No significant changes in the serum biochemical parameters were observed in the animals treated with the peptide P34. Signs of possible toxicity were no detected in animals in the group treated with 0.825 mg/kg day of peptide P34. In this group only histological changes in the spleen with the presence of megakaryocytes were observed. From these results show the potential o peptide P34 to be used in future as biopreservative in foods.

Higher eukaryotes produce a vast range of antimicrobial peptides (AMPs) that play important roles in their defence against microbial infection. Beta defensins are small (3-5 kDa), cationic peptides that display broad, potent antimicrobial activity against a range of microbes and also act as chemoattractants of important immunomodulatory cells. To generate highly pure peptides for structural and functional studies, we developed a method to prepare recombinant human beta defensin-2 (HBD2). The HBD2 gene was synthesised by recursive PCR with codons optimised for expression in Escherichia coli. HBD2 was expressed as an insoluble fusion to a His-tagged ketosteroid isomerase. After cleavage from the fusion with cyanogen bromide, 1H NMR spectroscopy and mass spectrometry confirmed that the oxidised HBD2 was folded and possessed the correct b-defensin disulfide bond topology. The recombinant HBD2 was active against E. coli, P. aeruginosa, S. aureus and C. albicans and was also a chemoattractant against HEK293 cells expressing the chemokine receptor CCR6. 15N-labelled HBD2 was also prepared and was highly suitable for future structural studies. Since defensins are thought to interact with bacterial membranes we also tested the recombinant HBD2 in biophysical studies (surface plasmon resonance, SPR, Biacore). We observed different binding to artificial model membranes containing either E. coli Kdo2-lipid A or phospholipids. Bacterial resistance to AMPs has been linked to the covalent modification of the outer membrane lipid A by 4-amino-4-deoxy-L-arabinose (L-Ara4N). This neutralises the charge of the LPS, thereby decreasing the electrostatic attraction of cationic peptides to the bacterial membrane. The pathogen Burkholderia cenocepacia displays extremely high resistance to AMPs and other antibiotics and the Ara4N pathway appears to be essential. To explore this further we expressed recombinant forms of two enzymes (ArnB and ArnG) from the B. cenocepacia Ara4N pathway. Purified ArnB is a pyridoxal 5’-phosphate (PLP)-dependent transaminase and we tested its ability to bind amino acid substrates. We investigated the binding of inhibitors L- and D-cycloserine to ArnB and tested their antibiotic activity against Burkholderia strains. We also studied the B. cenocepacia ArnG – a proposed membrane protein undecaprenyl-L-Ara4N flippase – and showed that the protein behaved as a dimer by non-denaturing gel analysis. The B. cenocepacia ArnG failed to complement E. coli knock-out strains encoding the equivalent flippase proteins ArnE and ArnF, suggesting that ArnG is a Burkholderia-specific protein.

2016 - 2017
Antimicrobial peptides (AMP) are evolutionarily conserved components of the innate immune system. They have a broad spectrum of action against bacteria, fungi and viruses. Therefore, AMP are studied as probable substitutes of the traditional antibiotics, for which most pathogens have developed resistance. The main objective of this work was the design of novel linear peptides capable to interact with the cellular membrane of the common pathogens. In this work, sequences of active AMP were carefully obtained from the scientific literature and collected in Yadamp (http://yadamp.unisa.it/), a database of AMP created recently in the laboratory where this project was carried out. In Yadamp, there are information about peptides name, amino acid sequence, length, presence of disulfide bridges, date of discovery, activity and taxonomy. The most relevant chemical-physical properties are also listed. This database is mainly focused on the peptides activities. Experimental MIC values (the lowest concentration of an antimicrobial that inhibits the visible growth of a microorganism) are constantly obtained from careful reading the original papers. In this work, a great contribution was made in the enrichment of the database. In fact, 1009 sequences were added to Yadamp. It currently contains 3142 AMP sequences. For these AMP, 573 molecular descriptors were calculated. In addition, this project also involved the search for new molecular descriptors. Yadamp is a resource for QSAR investigations on AMP. It allows to create subsets of AMP, homogeneous in one, two or more parameters...[abstract by Author]
XXX ciclo

This PhD thesis is the result of the combination of experimental and computational techniques with the aim of understanding the mechanism of action of de novo cyclic decapeptides with high antimicrobial activity. By experimental techniques the influence of the replacement of the phenylalanine for tryptophan residue in their antimicrobial activity was tested and the stability in human serum was also analyzed, in order to evaluate their potential therapeutic application as antitumor agents. On the other hand, the interaction amongst the peptide BPC194 c(KKLKKFKKLQ), the best candidate from the whole library of cyclic peptides, and a model anionic membrane was simulated. The results showed a structure-function relationship derived from the stable conformation of the peptides involved in the membrane permeabilization. As a result, a rational design was performed being BPC490 the peptide with best antimicrobial activity compared with the best active peptide from the original library.
Aquesta tesi doctoral resulta de la combinació d’estudis mitjançant tècniques experimentals i computacionals amb l’objectiu d’entendre el mecanisme d’acció de "de novo" decapèptids cíclics amb elevada activitat antimicrobiana. Experimentalment, es va avaluar la influència de la substitució dels residus de fenilalanina per triptòfan en la seva activitat antimicrobiana i també la seva estabilitat sèrum humà, per tal de valorar la seva possible aplicació terapèutica envers el càncer. Per altra banda, es va simular la interacció del pèptid BPC194 c(KKLKKFKKLQ), millor candidat de la biblioteca de pèptids cíclics, amb models aniònics de bicapa lipídica. Els resultats van posar en manifest una relació estructura-funció derivada de la conformació estable dels pèptids que participen directament en la permeabilització de la membrana. Es va procedir doncs al disseny racional de nous pèptids cíclics sent el pèptid BPC490 el que va presentar una millor activitat bacteriana en comparació amb el pèptid més actiu de la llibreria original.

Prosthetic-associated infections are one of the most challenging postoperative complications for orthopedic implants. The consequences that infections may lead to include patient pain, high cost, prolonged hospitalization time, and usually the revision of the implant. Current prophylaxis and therapy utilizing antibiotics are facing an emergency of increasing bacterial resistance; the design of a novel anti-infectious implant surface is therefore required. Among the potential antimicrobial alternatives are the antimicrobial peptides (AMP). AMPs are a family of natural defense peptides that has not received enough recognition until recently. The complex killing mechanisms of these cationic peptides make them very unlikely to encounter resistant mutants, and their broad-spectrum activity offers them great opportunity in possible clinical applications. In this study, a novel short AMP Tet213 with prominent bactericidal activity was chosen as the antimicrobial candidate and was covalently attached to titanium surfaces through a short bifunctional linker. This designed routine was confirmed with single cysteine before being applied to the 9-mer AMP candidate. The surface density of the immobilized AMP was determined by detecting its arginine residues after a reaction with 9,10-phenanthrequenon (PHQ). The reaction between arginine and PHQ generates a fluorescent product, by the emission of which the quantity of the arginine-containing peptide can be calculated. The density of the surface-attached Tet213 was measured to be 1.30±0.55 μg/cm². A relatively large proportion of physically adsorbed Tet213 was also observed, with the net adsorbed quantity to be 0.74±0.20 μg/cm². The affinity of the cationic AMP to the bare titanium surface is believed to be a result of electrostatic interactions. Both the covalently immobilized and the physically adsorbed Tet213 showed bactericidal activities of generally > 50% against a Pseudomonas aeruginosa (P. aeruginosa) strain which constitutively expresses luminescence when alive. The inhibition rate was calculated by the luminescence reduction and confirmed by the colony counts of the surviving bacteria. Several parameters were found to be influential to the overall inhibition rate, including the selection of the AMP candidate, the dilution of the bacterial culture and the bacterial incubation time.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2010.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Each year, 2 million people contract hospital-acquired bacterial infections, which causes the death of 100,000 patients and costs the US healthcare system over $21 billion. These infections have become dangerously resistant to our existing line of antibiotics and are rapidly spreading outside of hospitals and into communities. As molecular targets to develop new antibiotics are becoming exhausted, clinicians and scientist are concerned that antibiotic resistant infections will wipe out most of the major health benefits acquired over the last century. The work described in this thesis develops new antimicrobials strategies against bacterial infections, focusing on antimicrobial peptides (AmPs). We first delivered genes inducing the toxic expression of AmPs and other lytic agents directly into bacteria using re-engineered bacteriophages. Expression of these lytic agents in lysogenic bacteriophages resulted in bactericidal activity, and demonstrated, for the first time, a long-term cidal effect for over 20 hours. We then enhanced the efficacy of our approach by expressing the same agents in lytic bacteriophage, which resulted in complete suppression of the bacterial culture and prevented bacterial regrowth and resistance to bacteriophages. Since a large fraction of medical infections originates at the surface of implantable devices, we developed film coatings that release active AmPs to cover these surfaces and prevent bacterial colonization. We incorporated AmPs in layer-by-layer films and demonstrated that the kinetics of AmP release can be adjusted. These released AmPs still actively prevented bacterial growth and remained non-toxic towards mammalian cells. While natural AmPs have broad activity against pathogens, they are not optimized for a specific antimicrobial function or bacterial target. Thus, researchers have tried for decades to design highly active and specific de novo AmPs. One approach is to design new peptides using conserved motifs identified from the amino acid sequence of natural AmPs. We improved this approach by measuring the antimicrobial activity of a large database of natural AmPs and incorporating this activity information in the design algorithm. This strategy improved the success rate of designing de novo peptides from 45% to 73% and increased the antimicrobial strength of the designed peptides. Finally, we developed new potentiating strategies by studying the mode-of-action of the family of ponericin AmPs. First, we measured their cidal behavior and differentiated bactericidal ponericins from bacteriostatic ones. Using a modified AFM and a microfluidic device, we observed that the action of AmPs led to cellular death through the corrugation of bacterial, while subpopulation of cells resisted the action of the AmPs longer than others. Focusing on the ponericin G1 AmP, we correlated these visual observations with various membrane stress sensing mechanisms. We concluded that bacteria's ability to develop resistance to ponericin G1 requires the sensing and repair of misfolded membrane proteins via the CpxAR system, as well as DNA repair via induction of the SOS response by RecA. Using microarrarys, we showed that ponericin G1 targets tRNA synthetases in the ribosome. Finally, we demonstrated 99.999% killing of antibiotic resistant bacteria by potentiating ponericin G1 with the ribosomal antibiotic kanamycin, whereas no killing is observed when these two agents are applied independently. untreta The PhDCEP capstone requirement finalizes the work of this thesis by analyzing market entry and expansion strategies for an antimicrobial company commercializing genetically engineered bacteriophages. In conclusion, this thesis establishes new advances in the delivery, the design and the potentiation of AmPs in order to eradicate resilient bacterial infections.
by Tanguy Chau.
Ph.D.

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.

This master thesis project was carried out at SP Technical Research Institute of Sweden within the FORMAMP project which goal is to increase the efficiency and stability of antimicrobial peptides (AMPs) by exploring and developing a number of innovative formulation strategies for the drug delivery of those systems. In view of the growing problem of bacterial resistance to traditional antibiotics, AMPs represent one of the most promising alternatives as therapeutics against infectious diseases: besides having a fast and non-specific mechanism of action, they are less prone to bacterial resistance. In this project, the goal was to develop an efficient method for the formulation of hexagonal lyotropic phase nanodispersions (called hexosomes) as drug delivery vehicles for the AP114, DPK-060 and LL-37 AMPs. Then, these formulations were characterized through size measurements, zeta potential measurements, SAXS, cryo-TEM and UPLC and their stability was assessed. Lastly, the interaction of these systems with model bacterial membranes was tested through QCM-D and ellipsometry. The relevant samples were found to have a hexagonal structure with the lattice parameter being larger when peptide was loaded. The systems were observed to be sufficiently stable and the peptide loading efficiency was found to be higher than 90% in most cases. The hexosomes loaded with LL-37 were observed to preserve the effectiveness of the peptide when interacting with the model membrane through QCM-D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2007.
Includes bibliographical references (p. 248-268).
With the spread of drug-resistant bacteria, existing antibiotics are losing their potency. Antimicrobial peptides (AmPs) represent an exciting class of drug candidates, particularly because their mechanism of action is unlikely to induce drug resistance. If resistance to AmPs were also slower to emerge in the clinic, they would have longer useful lifetimes than existing antibiotics. Nevertheless, a number of limitations exist for AmPs in the clinic. The high cost of peptide manufacture requires that highly potent sequences are created. Additionally, AmP selectivity must be improved if effective systemic doses are to be given without hemolytic activity or other toxicity. Improved high-throughput methods for AmP design or discovery could enable the achievement of both of these goals. To this end, we developed an approach based on the discovery of semi-conserved motifs across natural AmPs, which we demonstrated are associated with antimicrobial activity. Additionally, we created novel AmP formulations that may bypass some of these clinical limitations. In order to evaluate AmP design approaches, a high-throughput production and assay platform was created using in vitro translation. This technology may produce peptides that would be toxic to recombinant hosts and synthesize peptides of arbitrary length.
(cont.) The cost per peptide was minimized through a series of process improvements. First, we created methods to construct oligonucleotides that mimicked our motif-based design of AmPs. This approach allowed the reuse of primers for many peptides, reducing cost and enabling the study of pattern synergy. Additionally, we found peptide translation was enhanced by co-translating a fusion partner in frame with the AmP. The AmP could be freed from the fusion partner after translation using enterokinase digestion. Further, we increased yield 3-fold by optimizing the length of fusion partner. The partner was made as short as possible to limit the translational resources required to synthesize the fusion partner, while being long enough to ensure stability from proteases. The solubility of the fusion partner-AmP construct was also improved through the selection of a highly soluble partner of the optimal length. Finally, we developed a purification scheme to ensure that the in vitro translation extract would not impact measurement of antimicrobial activity. We also developed and evaluated the design of AmPs using semi-conserved motifs. We used a database of over 500 natural AmPs as a training set for pattern discovery.
(cont.) The resulting motifs were exhaustively recombined to create all 20 amino acid sequences that were entirely covered by these patterns. These sequences were clustered, and 42 diverse members selected for characterization using representative Gram negative and Gram positive bacteria. Approximately 50% of the designed AmPs were active against at least one of the bacteria at 256 ug/ml. Control peptides were created in which the amino acids in the designed peptides were rearranged such that they were not homologous to any antimicrobial motifs. Thus, these controls had the same bulk physiochemical properties frequently associated with antimicrobial activity as the designed sequences, but we hypothesized they would not be active because they did not match the antimicrobial motifs. In fact, only 5% of the control sequences had activity at 256 ug/ml, indicating that the antimicrobial motifs give a 10-fold enrichment in activity. Further, two highly active designed peptides had MICs of 16 ug/ml against Bacillus cereus and 64 ug/ml against Escherichia coli. Additionally, AmPs active against B. cereus were all active against the hospital pathogen Staphylococcus aureus, and the bioterror agent, Bacillus anthracis.
(cont.) Our motif-based design may be most effective as the first stage of a two-stage design tool. In the first stage, highly diverse leads with novel profiles are created and evaluated. Promising leads could then be optimized using a variety of techniques. By creating just 44 variants of one lead, we designed an AmP with broad spectrum activity that had MICs of 16 ug/ml against E. coli and 8 ug/ml against B. cereus and 4 ug/ml against S. aureus. Another approach to build on our design tool would be to incorporate activity and toxicity characteristics of members of the training set into the design or scoring of new sequences. In order to begin assembling this data using a standardized method, a representative set of 100 natural, linear AmPs was chosen through clustering. Their antimicrobial activity against E. coli, S. epidermidis, and S. aureus were evaluated, along with hemolytic activity. When further supplemented, this information may enable an improved scoring metric to be created. Additionally, we systematically demonstrated that amidating the c-terminus of natural AmPs improves both antimicrobial activity and therapeutic index. Finally, we recognized that AmP's mechanism of action would allow activity to be retained when they are permanently tethered to medical device surfaces.
(cont.) Unlike existing coatings which rely on the slow release of silver or other antibiotics, a permanently tethered approach could have a longer lifetime and reduced systemic toxicity concerns. A versatile chemistry was developed to create immobilized AmP coatings. These formulations had broad spectrum antimicrobial activity without significant hemolytic activity. Further, the coatings were effective through multiple bacterial challenges. The combination of the AmP design tool along with localized formulations represent a significant advance in the process of moving AmPs to the clinic to combat drug-resistant infections.
by Christopher Loose.
Ph.D.

Despite many medical advances, antibiotic resistant bacteria increasingly plague the modern world, necessitating discovery of new antibiotics. One area of nature that can provide inspiration for antibiotics is antimicrobial peptides. Many of these peptides exist in nature, with some classes that have not been studied or characterized well. One such class is the defensin-like peptides generated by the plant Medicago truncatula as part of their symbiotic relationship with Sinorhizobium meliloti. Nodule-specific Cysteine Rich (NCR) peptides are defined by the presence of multiple cysteines, and regulate the growth of S. meliloti within plant cells. While some of these NCR peptides have been shown to have antimicrobial properties, hundreds of peptides remain uncharacterized. We have developed an assay for further characterization of these peptides in E. coli. Of the seven peptides that have been tested using this assay, three have exhibited definitive antimicrobial properties against both E. coli and S. meliloti. Additionally, we have developed a system for discovering novel antimicrobial peptides. This platform, called PepSeq, uses the expression of random peptides in E. coli combined with deep sequencing to detect antimicrobial activity. This technology is capable of screening through millions of peptide molecules simultaneously. Using this platform, we have discovered and confirmed six novel antimicrobial peptides, with hundreds of additional predicted antimicrobial peptides. In addition to the peptides we have analyzed using PepSeq, additional peptide scaffolds could be used to discover more potent antimicrobial peptides.

The emergence of multidrug resistant strains of bacteria has resulted in the need for novel therapeutic agents. The ApoEdp peptide, derived from the receptor-binding region of the human apolipoprotein E, had previously been shown to have activity against herpes simplex viruses, human immunodeficiency virus and certain bacterial species. However, its antibacterial mode of action was not elucidated, therefore the present study aimed to determine this mechanism. The susceptibility of several different strains, including Pseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium smegmatis, Staphylococcus epidermidis and Escherichia coli, to ApoEdp was investigated. No significant difference was observed between the minimal inhibitory concentrations (MICs) of ApoEdp against a range of Gram positive and Gram negative bacteria. The presence of E. coli K5 capsular polysaccharide in the growth medium led to a decrease in ApoEdp susceptibility of the non-capsulated E. coli MS101 DeltakfiC strain. Bacteria with non-functioning multidrug efflux pumps showed no difference in susceptibility. A mutation in the phoP gene of Salmonella enterica Serovar Typhimurium LT2, which regulates cell surface modifications led to an increase in ApoEdp susceptibility. Transmission electron microscopy (TEM) images showed changes in the membrane and internal structures of strains incubated with a minimal bactericidal concentration (MBC) of ApoEdp for 5 min. ApoEdp was able to depolarise the cytoplasmic membrane. The ability of ApoEdp to induce cell lysis was assessed by the release of β-galactosidase into the supernatant. There was no significant difference in the supernatant β-galactosidase levels of ApoEdp treated and unlysed cells. ApoEdp, however was able to form pores in artificial lipid bilayers and decrease intracellular ATP levels. The effect of ApoEdp on transcription and translation was determined using an in vitro transcription/translation system. Results showed that ApoEdp did not affect protein synthesis. ApoEdp also worked in synergy with rifampicin, chloramphenicol, ampicillin and ciprofloxacin against bacteria. Overall, the results showed that ApoEdp acts by targeting the cytoplasmic membrane, although it may also have intracellular targets. Its ability to work in combination with conventional antibiotics and antibacterial activity against a range of different bacteria species demonstrates its therapeutic potential.

Thesis advisor: Jianmin Gao
As the threat of microbial resistance to antibiotics grows, we must turn in new directions to find new drugs effective against resistant infections. Antimicrobial peptides (AMPs) and host-defense peptides (HDPs) are a class of natural products that have been well-studied towards this goal, though very few have found success clinically. However, as there is much known about the behavior of these peptides, work has been done to manipulate their sequences and structures in the search for more drug-like properties. Additionally, novel sequences and structures mimicking those seen in nature have been discovered and characterized. Herein, we demonstrate our ability to finely tune the antimicrobial activity of various peptides, such that they can be provided with more clinically desirable characteristics. Our results show that gramicidin A (gA) can be made to be less toxic via incorporation of unnatural cationic amino acids. This is achieved by synthesizing lysine analogues with diverse hydrophobic groups alkylated to the side-chain amine. Through exploring different groups, we achieved peptide structures with improved selectivity for bacterial over mammalian membranes. Additionally, we were able to achieve novel broad-spectrum gram-negative activity for gA peptides. In efforts to combat bacterial resistance to cationic antimicrobial peptides (CAMPs), we have directed our reported amine-targeting iminoboronate chemistry towards neutralizing Lys-PG in bacterial membranes. Originally incorporating 2-APBA into gA, we found this to hinder the peptide’s activity. However, we were successful in increasing the potency of gA3R, a cationic mutant of gA, towards S. aureus by using a co-treatment of this peptide with a Lys-PG binding structure. Currently, we are exploring this strategy further. Finally, we describe our work towards establishing a novel cyclic peptide library incorporating a 2-APBA warhead for iminoboronate formation with a given target. In this, we have achieved intermolecular reduction of iminoboronates, strengthening the stringency of library screening. Although we were unsuccessful in finding a potent hit for binding of the lipid II stem peptide, screening against human transferrin yielded selective hits. Currently we are investigating these hits to understand their activity and therapeutic potential
Thesis (PhD) — Boston College, 2017
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

In the present PhD thesis we studied the solid-phase peptide synthesis of antimicrobial peptides derived from the lead peptides BP100 and BPC194. First, peptides derived from BP100 containing D-amino acids at different positions of the sequences were prepared. Moreover, peptidotriazoles derived from BP100 were also synthesized containing the triazole ring at the side-chain of different amino acids. Then, we proceeded to perform studies for the synthesis of multivalent peptides derived from BPC194. To achieve this objective, the synthesis of cyclic peptides containig a triazole ring at amino acids side-chain with different elongations was carried out. Finally, we prepared various carbopeptides containing 2 and 4 units of BP100 and/or its derivatives. The evaluation of the biological activity allowed the identification of active sequences against the economically important phytopathogenic bacteria and fungi and not toxic against eukaryotic cells.
En aquesta tesi doctoral es va estudiar la preparació en fase sòlida de pèptids antimicrobians derivats dels pèptids lead BP100 i BPC194. En primer lloc, es varen preparar derivats del pèptid lineal BP100 incorporant D aminoàcids en diferents posicions de la seqüència. A més, també es varen sintetitzar derivats d'aquest pèptid lead incorporant un anell de triazole a la cadena lateral de diferents aminoàcids. Posteriorment, es va procedir a l'estudi de la síntesi dd pèptids multivalents derivats de BPC194. Per aconseguir aquest objectiu es va portar a terme la síntesi de pèptids cíclics incorporant un anell de triazole a la cadena lateral d’aminoàcids amb diferents allargades de cadena. Finalment, va procedir a la preparació de carbopèptids contenint 2 i 4 unitats de BP100 i/o derivats. L’avaluació de l’activitat biològica dels pèptids sintetitzats va permetre idenficar seqüències actives enfront de bacteris fitopatògens i fongs i poc tòxiques enfront cèl•lules eucariotes.

With the culture of marine fish species increasing, interest in ways to combat infection in these species has grown. One such avenue is the search for novel antimicrobials. Antimicrobial peptides (AMPs) are host derived molecules with a significant role in innate immune responses and have been studied extensively in mammals. Well known AMP families include β-defensins, cathelicidins, liver expressed antimicrobial peptides and, specific to fish, the piscidins. While these molecules have been found in some fish species mining of expressed sequence tag (EST) databases was carried out to identify them in other fishes. In this study we have found new members of the β-defensin, cathelicidin and piscidin families of AMPs, while also identifying new hepcidins and LEAP-2 molecules in a diverse range of fish species. The rest of this thesis focused on one major AMP family, the cathelicidins, which in fish have been mainly identified in salmonids. They are stored in secondary granules of neutrophils as inactive pro-peptides and when infection occurs, a cleavage event releases the active mature form that effects microbicidal activity. Characterization of the cathelicidin genes present in Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) was undertaken. A number of ESTs with significant homology to cathelicidins were found in Atlantic cod. Primers were designed to amplify these sequences by PCR in both gadoid species, and the gene products were cloned and sequenced. One and two cathelicidin genes were identified in cod (gmCath1) and haddock (maCath1; maCath2), respectively, which differ in the length of their active mature peptides. Gadoid cathelicidins were constitutively expressed in many tissues and were upregulated by a range of immunostimulants. Analysis of the gmCath1 promoter revealed numerous putative transcription factor binding sites, some of which were used in an expression vector construct to show upregulation of gene expression when stimulated with the viral RNA mimic Poly I:C. Synthetic and recombinant gadoid cathelicidins were produced but did not appear to display antimicrobial activity, so this requires further investigation. The discovery of new members of the major AMP families in such a diverse range of fish species bodes well for future research into the use of these antimicrobials as potential disease control molecules in the aquaculture industry. Particularly for Atlantic cod this is crucial as the major cod farms in Norway are experiencing difficulties with bacterial infections for which there is no vaccine at present.

Antimicrobial resistance is a challenge facing the world in the twenty-first century with an estimated 10 million deaths by 2050 if no actions are taken. Microbial resistance to drugs is a natural consequence when bacteria develop and adapt genetically to face new challenges including antibiotics. Currently, this development occurs at a higher rate than drug discovery. Hence there is a need for a new generation of antibiotics that kill pathogenic bacteria. Nature itself provides inspiration for such new antibiotics. For example, our immune system secretes antimicrobial peptides (AMPs), which have been successful agents in killing pathogens with no reported bacterial resistance. Compared with conventional antibiotics, these peptides are larger and more sophisticated biological molecules, which disturb the bacterial membrane, leading to cell lysis. It is currently costly to extract AMPs from natural resources to be used for fighting infections. Alternatively, synthetic AMPs that mimic natural ones could provide a sustainable cheap weapon against such thread. This also provides a unique opportunity to understand the structure–function relationships of such molecules to optimise these effective, non-toxic antimicrobial properties. Our collaborators at National Physical Laboratory have designed and synthesised new AMPs from their essential building blocks (amino acids). This thesis describes the use of atomic force microscopy (AFM) as a nanoscale imaging technique for characterising and imaging membrane poration mechanisms of four new AMP systems. Two of these systems are helical peptides, explained in chapter 3. The third system, explained chapter 4, is a triskelion with three arms of antimicrobial β-sheet peptide that co-assemble to form a hollow antimicrobial capsules. The latter has two possible functions: gene delivery and bactericidal effects. The fourth system, explained in chapter 5, contains two peptide monomers that are designed to co-assemble and form antimicrobial hollow capsids, inspired by the natural viral capsids. Finally, chapter 6 is a plan for taking these AMPs a step closer to commercialisation, including a business plan for one potential application.

Clostridium difficile (CD) is the leading cause of bacterial hospital-associated infection in North America. How CD colonizes the human host, including its response to the innate immune system and other stresses, is poorly understood. This work considers CD's defenses against two stresses found in the host - the antimicrobial peptide LL-37 and reactive oxygen species (ROS). LL-37 had bactericidal activity against CD. CD strains varied in their sensitivity to the peptide, and epidemic-associated strains were more resistant to LL-37 than others. CD became more resistant to LL-37 following exposure to sub-lethal concentrations of the peptide, suggesting the presence of inducible resistance mechanisms. A quantitative proteomics analysis revealed definite alterations in CD protein expression caused by LL-37. Specific changes included increased expression of DltB, a protein previously reported to confer resistance against other antimicrobial peptides. Notably, disruption of individual LL-37-induced genes did not sensitize CD to the peptide. This suggests functional redundancy, and that LL-37 may cause global changes in protein expression, not limited to antimicrobial peptide resistance determinants. One of the proteins most strongly induced by LL-37 was a predicted superoxide reductase (SOR). As CD is considered a strict anaerobe, expression of a predicted antioxidant protein was an interesting finding. Heterologous expression of CD SOR in a superoxide dismutase-deficient E. coli strain confirmed its action as a superoxide scavenger. Insertional inactivation of SOR rendered CD more sensitive to oxygen and ROS-generating compounds, indicating that SOR contributes to antioxidant defense in CD. SOR mutants were impaired in their ability to cause disease in hamsters, indicating a role for this protein in infection.

Thesis (MSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Biofouling is the attachment and biofilm formation that leads to negative repercussions such as persistent post-harvest infections, infections obtained from medical implants and continual surface contamination of food processing plants. Much of the problem lies with the resistance that develops against conventional treatments due to the formation of mature biofilms. Thus the focus has shifted from the removal of biofilms to the prevention of initial attachment of organisms. This entails the use of antimicrobial surfaces that either have an inherent antimicrobial activity, e.g. certain metals, or surfaces that are modified by the attachment of antimicrobial agents. The attachment of antimicrobial agents can either be through covalent bonding or adsorption, depending on the intended use of the surface as well as the mode of action of the antimicrobial agent. Antimicrobial peptides (AMPs) are ubiquitous in nature, tend to have a broad spectrum of activity, are very stable and have been shown to maintain activity when covalently bound to solid surfaces. Tyrocidines (Trcs), antimicrobial peptides produced by Bacillus aneurinolyticus, are cyclodecapeptides with a broad spectrum of activity against Grampositive bacteria, fungi, yeasts and the human malaria parasite, Plasmodium falciparum. The aim of this study was to determine the antimicrobial activity of surfaces treated with a tyrocidine extract, under which conditions the activity remained stable and to look into possible applications of these peptide-treated surfaces. The study focussed on different solid surfaces namely mixed cellulose, polyvinylidene fluoride, polycarbonate, cellulose acetate, cellulose (paper)(CL) and high density cellulose packing material (HDC), as a pilot study to assess the antimicrobial activity of Trc and gramicidin S (GS) treated solid surfaces. Peptide desorption and subsequent analysis by mass spectrometry was used to confirm the presence and integrity of the Trcs adsorbed. Scanning electron microscopy was utilised to show that the adsorbed peptides did not affect the structural integrity of the treated filters. However, it was shown that the adsorbed peptides changed the hydrophobic/hydrophilic character by means of a wettability assay. A cell viability assay and erythrocyte assay were developed from existing methodologies to determine the biological activity of the AMP-functionalised polymeric material. Seven of the AMP treated solid surfaces showed antimicrobial activity when challenged with >105 Micrococcus luteus cells/cm2. Although the polycarbonate filter lost antimicrobial activity at the high cell concentrations, it was shown to have potent antimicrobial activity at lower cell concentrations. Complete inhibition of M. luteus growth was observed for both the gramicidin S and tyrocidine extract treated high density cellulose and cellulose filters. Stability tests showed that the tyrocidines remained adsorbed to cellulose filters and biologically active when exposed multiple water washes, water washes at different temperatures (25°C - 100°C) and pH changes (pH 1-12). The antimicrobial activity was only affected after exposure to the water wash of pH 13 which is possible due to susceptibility of the CL filters to high pH solvents. A preliminary study on the effect of Trcs treated CL filters on the sterilization, germination and effect on tomato seedlings was conducted. It was found that Trcs had no effect on the germination and did not fully sterilise the seeds or environment against fungi. However, it was observed that 5 μg/mL Trcs treated filters promoted root length opposed to the toxic effect seen with filters treated with higher Trc concentrations. It is hypothesised that Trcs prefer to bind to hydrophilic surfaces exposing the hydrophobic residues and the cationic residue of the peptide to interact with the bacterial membrane to elicit its antimicrobial response. The exposed residues contain some of the hydrophobic residues and the cationic Orn9/Lys9, which are crucial to the antimicrobial activity of the peptides. Hydrophobic interaction is particularly important for the haemolytic activity which is currently the only viable method of detection of the adsorbed Trcs. Trcs also have a preference for adsorption onto cellulose and cellulose analogues which points to possible application in protective food wrapping and wood surface protection. Trcs maintains its antimicrobial activity regardless of adsorption to solid surfaces. It can therefore be concluded that Trcs treated solid surfaces hold great potential in preventing the initial bacterial colonization and subsequent biofilm formation. Antimicrobial peptide enriched solid surfaces can thus be developed and tailored to a specific application such as filters, catheters and packaging materials.
AFRIKAANSE OPSOMMING: Biovervuiling is die aanhegting en vorming van biofilms met negatiewe gevolge soos aanhoudende na-oes infeksies, infeksies op mediese inplantings en voortdurende oppervlak besoedeling van voedselverwerkings fabrieke. Die probleem lê grotendeels by die weerstand wat ontwikkel word teen konvensionele behandelings as gevolg van die vorming van volwasse biofilms. Die fokus het gevolglik verskuif vanaf die verwydering van biofilms na die voorkoming van aanvanklike aanhegting van organismes aan oppervlaktes. Dit behels die gebruik van antimikrobiese oppervlaktes wat of 'n inherente antimikrobiese aktiwiteit het, bv. sekere metale óf oppervlaktes wat aangepas is deur die aanhegting van antimikrobiese middels. Die aanhegting van antimikrobiese agente kan of deur kovalente binding óf adsorpsie plaasvind, afhangende van die beoogde gebruik van die oppervlak, sowel as die metode van werking van die antimikrobiese agent. Antimikrobiese peptiede (AMPe) is alomteenwoordig in die natuur, is geneig om 'n breë spektrum van aktiwiteit te hê, is baie stabiel en het getoon dat aktiwiteit in stand gehou word wanneer dit kovalent gebind word op soliede oppervlaktes. Tirosidiene (Trcs), antimikrobiese peptiede wat deur Bacillus aneurinolyticus geproduseer word, is siklodekapeptiede met 'n breë spektrum van aktiwiteit teen Gram-positiewe bakterieë, swamme, giste en die menslike malaria parasiet Plasmodium falciparum. Die doel van hierdie studie was om die antimikrobiese aktiwiteit te bepaal van oppervlaktes wat met 'n tirosidien ekstrak behandel is, te bepaal onder watter omstandighede die aktiwiteit stabiel bly en om te soek na moontlike toepassings van hierdie peptied-behandelde oppervlaktes. Die studie het gefokus op verskillende soliede oppervlaktes naamlik gemengde sellulose, polyvinylidene fluoried, polikarbonaat, sellulose asetaat, sellulose (papier)(CL) en 'n hoë digtheid sellulose verpakkings materiaal (HDC), as 'n loodsstudie om die antimikrobiese aktiwiteit van die Trcs en gramisidien S (GS) behandelde soliede oppervlaktes te ondersoek. Peptied-desorpsie en daaropvolgende ontleding deur massaspektroskopie is gebruik om die teenwoordigheid en integriteit van die geadsorbeerde Trcs te bevestig. Skandering elektronmikroskopie is gebruik om aan te toon dat die geadsorbeerde peptiede geen invloed op die strukturele integriteit van die behandelde filters het nie. Daar is egter getoon dat die geadsorbeerde peptiede die hidrofobiese / hidrofiliese karakter verander. „n Lewensvatbaarheid selgebaseerde toets en eritrosiet toets is ontwikkel uit bestaande metodes om die biologiese aktiwiteit van die AMP-gefunktionaliseerde polimeriese materiaal te bepaal. Sewe van die AMP behandel soliede oppervlaktes het antimikrobiese aktiwiteit getoon wanneer dit met > 105 Micrococcus luteus selle/cm2 gedaag is. Hoewel die polikarbonaat filter antimikrobiese aktiwiteit met hoë sel konsentrasies verloor het, is dit getoon dat dit wel uitgeproke antimikrobiese aktiwiteit het teen laer konsentrasies selle. Volledige inhibisie van M. luteus groei is waargeneem vir beide die hoë digtheid sellulose en sellulose filters wat met GS en tirosidien ekstrak behandel is. Stabiliteit toetse het getoon dat die tirosidiene geadsorbeer en biologies aktief op sellulose filters bly nadat dit blootgestel is aan verskeie water was-stappe, waterwasse by verskillende temperature (25 °C -100 °C) en pH veranderinge (pH 1-12). Die antimikrobiese aktiwiteit was net beïnvloed ná blootstelling aan die water met 'n pH 13, wat moontlik is te danke aan die vatbaarheid van die CL filters by hoë pH oplosmiddels is. 'n Voorlopige studie is gedoen om die uitwerking van Trcs behandelde CL filters op die sterilisasie, ontkieming en tamatiesaailinge te bepaal. Daar is gevind dat Trcs geen effek op die ontkieming het nie, maar dat dit nie volledig die sade en omgewing steriliseer vir fungiese groei nie. Daar is egter waargeneem dat 5 μg/mL Trcs behandelde filters wortel lengte van die saailinge bevorder teenoor die giftige uitwerking soos waargeneem vir die filters wat met hoër konsentrasies Trcs behandel is. Dit word gepostuleer dat Trcs verkies om aan hidrofiliese oppervlaktes te bind wat die van die hidrofobiese aminosure en die kationiese residu van die peptied blootstel om aan die bakteriële membraan te bind om gevolglik antimikrobiese reaksie te ontlok. Die blootgestelde deel bevat sommige van die hidrofobiese residue en positiewe Orn9/Lys9 wat noodsaaklik vir die antimikrobiese aktiwiteit van die peptiede. Die hidrofobiese interaksies is veral belangrik vir die hemolitiese aktiwiteit wat tans die enigste bruikbare metode van opsporing van die geadsorbeerde Trcs is. Trcs het ook 'n tendens vir adsorpsie op sellulose en sellulose analoë wat dui op die moontlike toepassing in beskermende voedselverpakking en die beskerming van houtoppervlaktes. Trcs handhaaf hul antimikrobiese aktiwiteit, ongeag van adsorpsie aan soliede oppervlaktes. Dit kan dus afgelei word dat Trcs-behandelde soliede oppervlaktes die potensiaal het om die aanvanklike kolonisasie van bakterië te voorkom en die daaropvolgende biofilm vorming. Antimikrobiese peptied verrykde soliede oppervlaktes kan dus ontwikkel en aangepas word vir gebruik in spesifieke toepassing soos in filters, kateters en verpakkingsmateriaal.

The rise in antimicrobial resistance and paucity of new antimicrobial compounds calls for alternatives to traditional antibiotics. Antimicrobial peptides (AMPs) have emerged as potential candidates. Cathelicidins are a major family of AMPs in mammals which form part of innate immunity through antimicrobial and immunomodulatory functions. Marsupial and monotreme cathelicidins are of particular interest due to their involvement in protecting immunologically naive young during development in the pouch via expression in the pouch lining and milk where they modulate microbial flora and provide passive immunity. As such, the cathelicidin gene family has expanded in marsupials and monotremes, with a high number of cathelicidins in the tammar wallaby, gray short-tailed opossum and platypus. However our knowledge is limited to these species and functional studies involving antimicrobial activity are lacking. This thesis describes the characterisation of cathelicidins in the Tasmanian devil, koala and echidna, and investigates the antimicrobial function of all marsupial and monotreme cathelicidins. As expected, cathelicidins have expanded in the Tasmanian devil and koala, resulting in a high number of cathelicidins which were widely expressed throughout the body, including in pouch lining and milk. Only a single cathelicidin was identified in the echidna due to the quality of the genome. Out of 26 cathelicidins tested, six displayed broad-spectrum antibacterial activity against gram-negative and positive bacteria, including methicillin-resistant Staphylococcus aureus. One koala cathelicidin rapidly inactivated C. pecorum and significantly reduced the number of chlamydial inclusions in vitro. Activity was reduced in the presence of serum and whole blood, and peptides displayed varying levels of haemolytic and cytotoxic activity. Many cathelicidins did not display antimicrobial activity and future work is required to explore their potential immunomodulatory properties. The results presented in this thesis have advanced our understanding of cathelicidins in marsupials and monotremes on a genetic and functional level, and highlights their potential as novel therapeutics in the future.

The incorporation of unsymmetrical biaryl systems into peptide sequences is a strategy that can improve their biological activity. Due to the difficulty of arylating the 4(5}-position of the imidazole ring, this doctoral thesis was focused on the development of efficient methodologies for the solid-phase synthesis of 5-arylhistidine-containing antimicrobial undecapeptides through a Suzuki-Miyaura reaction under microwave irradiation. The extension of this protocol allowed the preparation of biaryl cyclic peptides of different ring sizes bearing a His-Phe or His-Tyr biaryl linkage. Then, it was developed a procedure for the total solid-phase synthesis of biaryl cyclic lipopeptides derived from arylomycins. These strategies were extended to the preparation of biaryl cyclic analogues of the marine bicyclic pptides aciculitins. In particular, it was achieved the synthesis of analogues of the northern and the southern hemispheres of aciculitins as well as biaryl bicyclic peptides incorporating a Phe-Phe, a Phe-Tyr, a His-Tyr or a Tyr-Tyr biaryl bridge
La incorporació de sistemes biarílics asimiètrics en seqüències peptídiques es considera un enfocament útil per a millorar l'activitat biològica de pèptids. Tenint en compte la dificultat d'arilar la posició 4 (5) de l'anell d'imidazole, aquesta tesi doctoral es centra en el desenvolupament de noves estratègies eficients per a la preparació en fase sòlida d'undecapèptids antimicrobians contenint una 5-arilhistidina a través d'una reacció de Suzuki-Miyaura sota irradiació microones. L'extensió d'aquesta metodologia ha permès la síntesi de pèptids biarílics cíclics de diferents mides que incorporen un enllaç His-Phe 0 His-Tyr. Posteriorment, s'ha desenvolupat un procediment per la síntesi total en fase sòlida de lipopèptids biarílics cíclics derivats de les arilomicines. Les estratègies anteriors s'han estès a la preparació de compostos biarílics anàlegs dels pèptids bicíclics marins aciculitines. Concretament, s'ha preparat anàlegs dels hemisferis nord i sud de las aciculitines així com pèptids biarílics bicíclics que incorporen un pont Phe-Phe, Phe-Tyr, Tyr-His 0 Tyr-Tyr.

A series of antimicrobial peptides (AMPs) derived from the BP134 peptide (a member of the CECMEL11 library) exhibited potent bactericidal effect and low cytotoxicity, and were selected for expression in transgenic rice. Transgenic plants were generated expressing a codon-optimized AMP, under the control of a seed-specific promoter. It was confirmed that the expected peptides accumulated in the transgenic rice seeds, but not in other plant tissues, resulting in enhanced levels of protection against bacterial and fungal plant pathogens. Procedures for purification of the AMPs from seeds have been established based on solubilization/selective precipitation and Weak Cation Exchange-Reverse Phase chromatography. One of the peptides obtained, the S-Cec A, exhibited a high antibacterial and bactericidal activity against Dickeya sp. The accumulation levels of the recombinant AMP peptides in rice seeds were dependent on the strategy used, and its production in seeds offers a promising perspective for the use of rice as biofactory
Una sèrie de pèptids antimicrobians (PAMs) derivats del pèptid BP134 (provinent de la quimioteca CECMEL11), que exhibien un potent efecte bactericida i baixa citotoxicitat, es van seleccionar per a l'expressió en arròs. Es van generar plantes transgèniques que expressaven les seqüències gèniques corresponents als PAMs, i adaptades a l’ús de codons, sota el control de promotors específics de llavor. Es va confirmar que els PAMs esperats s’acumulaven en les llavors, però no en altres teixits de la planta, i conferien una protecció molt significativa a la infecció per patògens fúngics i bacterians de plantes. La purificació dels PAMs es va realitzar mitjançant solubilització/precipitació selectiva/ i cromatografia de Bescanvi Catiònic Feble i Fase Reversa. Un dels pèptids obtinguts, S-Cec A, exhibia una potent activitat antibacteriana i bactericida contra Dickeya sp. Els nivells d'acumulació dels PAMs en les llavors eren dependents de l'estratègia utilitzada. La llavor d’arròs ofereix una plataforma adequada pel seu ús com a biofactoria de PAMs

Antimicrobial peptides (AMPs) play a key role in the innate immune response. They can be ubiquitously found in a wide range of eukaryotes including mammals, amphibians, insects, plants, and protozoa. In lower organisms, AMPs function merely as antibiotics by permeabilizing cell membranes and lysing invading microbes. Prediction of antimicrobial peptides is important because experimental methods used in characterizing AMPs are costly, time consuming and resource intensive and identification of AMPs in insects can serve as a template for the design of novel antibiotic. In order to fulfil this, firstly, data on antimicrobial peptides is extracted from UniProt, manually curated and stored into a centralized database called dragon antimicrobial peptide database (DAMPD). Secondly, based on the curated data, models to predict antimicrobial peptides are created using support vector machine with optimized hyperparameters. In particular, global optimization methods such as grid search, pattern search and derivative-free methods are utilised to optimize the SVM hyperparameters. These models are useful in characterizing unknown antimicrobial peptides. Finally, a webserver is created that will be used to predict antimicrobial peptides in haemotophagous insects such as Glossina morsitan and Anopheles gambiae.

The rapid emergence of bacterial resistance to antibiotics has necessitated the development of alternative treatment strategies. Antimicrobial peptides (AMPs) are important immune system components that kill microbes rapidly and have broad activity-spectra, making them promising leads for new pharmaceuticals. Although the need for novel antimicrobials is great, we also need a better understanding of the mechanisms underlying resistance development to enable design of more efficient drugs and reduce the rate of resistance development. The focus of this thesis has been to examine development of bacterial resistance to AMPs and the resulting effects on bacterial physiology. The major model organism used was Salmonella enterica variant Typhimurium LT2. In Paper I, we observed that bacteria resistant to PR-39 appeared at a high rate, and that the underlying sbmA resistance mutations were low cost or even cost-free. Such mutants are more likely to rapidly appear in a population and, most importantly, will not disappear easily once the selective pressure is removed. In paper II, we isolated protamine-resistant hem- and cydC-mutants that had reduced growth rates and were cross-resistant to several other antimicrobials. These mutants were small colony variants (SCVs), a phenotype often associated with persistent infections. One SCV with a hemC-mutation reverted to faster growth when evolved in the absence of protamine. In paper III, the mechanism behind this fitness compensation was determined, and was found to occur through hemC gene amplification and subsequent point mutations. The study provides a novel mechanism for reversion of the SCV-phenotype and further evidence that gene amplification is a common adaptive mechanism in bacteria. In Paper IV, the antibacterial properties of cyclotides, cyclic mini-proteins from plants, were evaluated. Cycloviolacin O2 from violets was found to be bactericidal against Gram-negative bacteria. Cyclotides are very stable molecules and may be potential starting points for development of peptide antibiotics.

Le ribosome bactérien (70S) catalyse la formation de la liaison peptidique et représente une cible majeure pour les antibiotiques. Le peptide synthétisé passe à travers le tunnel de sortie de la sous-unité 50S du ribosome avant d’être libéré dans le cytoplasme. Des peptides spécifiques peuvent inhiber la traduction en agissant en cis (peptides naissants) ou en trans (peptides antimicrobiens riches en proline, PrAMPs) sur ce tunnel. Il a été montré que les PrAMPs inhibent la synthèse des protéines en se liant au ribosome 70S. Au cours de ma thèse, j’ai résolu les structures cristallines de quatre PrAMPs en complexe avec le ribosome 70S. J’ai ainsi pu révéler que tous ces peptides recouvrent le centre peptidyl transferase (PTC) et se lient avec le tunnel dans une orientation inverse par rapport au peptide naissant. J’ai aussi pu conclure que les PrAMPs inhibent la traduction en bloquant la transition de la phase d’initiation vers l'élongation. L'arrêt de la traduction induit par le peptide naissant se produit lorsqu'un peptide naissant interagit avec le tunnel, entraînant l'inactivation du PTC. L'arrêt peut être uniquement dû à la séquence du peptide ou peut nécessiter un co-inducteur, tel un antibiotique. Les mécanismes d'action des peptides d'arrêt courts (motifs polyproline ou M+X(+)) restent inconnus. Afin d'étudier ces peptides de manière biochimique et structurale, j’ai formé des complexes ribosomaux bloqués avec un peptidyl-ARNt d'arrêt préparé à l'aide d'un ribozyme appelé flexizyme. J’ai ainsi pu obtenir une structure par cryo-EM d’un 70S bloqué par un motif M+X(+) en présence d'érythromycine et de formuler un modèle expliquant l'inactivation allostérique du PTC
The bacterial (70S) ribosome catalyzes peptide bond formation and represents a major target for antibiotics. The synthesized peptide passes through the exit tunnel of the large ribosomal subunit before it is released into the cytoplasm. Specific peptides can inhibit translation by acting in cis (nascent peptide) or in trans (proline-rich antimicrobial peptides; PrAMPs) due to interactions with the tunnel. PrAMPs were reported to inhibit protein biosynthesis and bind to the 70S ribosome. During my thesis, I solved the crystal structures of four different PrAMPs in complex with the bacterial ribosome, revealing that all peptides cover the peptidyl transferase center (PTC) and bind in a reverse orientation within the exit tunnel relative to a nascent chain. From this, I concluded that PrAMP binding inhibits the transition from initiation towards elongation. Nascent chain-mediated translational arrest occurs when a nascent peptide interacts with the exit tunnel, leading to the rearrangement and inactivation of the PTC. Arrest can be solely due to the peptide’s sequence or may require a small molecule co-inducer, such as a drug. The underlying mechanisms of action for short arrest peptides (polyproline or M+X(+) motifs) remain unknown. In order to study these short arrest peptides biochemically and structurally, I adopted a strategy to form arrested ribosomal complexes through the direct addition of the arrest peptidyl moiety to tRNAiMet with the help of a small ribozyme known as flexizyme. I was able to solve the cryo-EM structure of a ribosome arrested by an M+X(+) motif in the presence of erythromycin and to propose a model for the allosteric inactivation of the PTC

MRSA is a significant pathogen, which can cause a range of minor and major infections both in the hospital and community environments. MRSA is developing resistance to many antibiotics, including vancomycin, which is now the first choice antibiotic to treat MRSA infections in the UK. This together with the dearth of new antibiotics being introduced could see the emergence of untreatable S. aureus strains. This has led to renewed interest in alternative antimicrobial agents. Lysostaphin is an endopeptidase produced by Staphylococcus simulans biovar staphylolyticus, which cleaves the peptidoglycan cross-bridges of other staphylococcal species. Lysostaphin has been investigated as a potential therapeutic agent and has shown promise in in vitro and in vivo studies and in clinical trials. However, resistance to lysostaphin is likely to emerge and there will be a demand for second generation Iysostaphins and/or other similar novel antimicrobials that can counteract this resistance. This study describes the cloning, purification and assaying of an endolysin of the S. aureus P68 bacteriophage. Lys16 lysin has previously been shown to possess staphylolytic activity. This study demonstrates that the purified recombinant protein is poorly soluble and is inactive against live cells. The Atl autolysin of S. aureus was also investigated as a potential antimicrobial. This study confirmed the hydrolytic profiles of the enzymes, and a chimeric peptide incorporating the lysostaphin targeting domain with the Atl glucosaminidase was designed. This did not confer greater activity against S. aureus, although the targeting domains of each enzyme were shown to utilise different cell surface receptors. Finally, this study reports the development of a novel assay to measure the activity of antimicrobial peptides against S. aureus, using a bioluminescence reporter. This was shown to be a sensitive assay, able to distinguish small differences in the activity of antimicrobial peptides.

This thesis initially describes the quest for sequence selective DNA followed by the search for new antimicrobial compounds. The first four chapters are based on DNA recognition with new cancer therapeutics being the long term goal, while the remaining three focus on finding new drugs for bacteria.

The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development. In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4. The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways. Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host. In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.

Rapid development of antimicrobial resistance (AMR) among bacteria, combined with diminished new antibiotic discovery rates, is an increasing threat to human health. Bacterially derived antimicrobial peptides (AMP) hold excellent potential as potent novel therapeutics. This study embraces traditional natural AMP discovery methods and the newer in silico genome mining tool BAGEL 3 to facilitate identification of novel antimicrobial agents. The traditional screening efforts led to the discovery of two promising antimicrobial producer strains; Bacillus pumilus J1 producing two AMPs, peptides NI03 and NI04, and Klebsiella pneumoniae A7, which produced peptide NI05. In silico mining of the B. pumilus J1 and K. pneumoniae A7 genomes and those from under exploited anaerobic bacteria using BAGEL 3 yielded 18 putative bacteriocin structures that were associated with multiple known and relevant bacteriocin accessory genes and/or carried significant homologies to known bacteriocins. Peptide NI04 proved to be active against Gram positive species only, including meticillin resistant Staphylococcus aureus and vancomycin resistant enterococci and peptide NI03, in addition to these pathogens, showed activity against E. coli. Peptide NI05 was active against Gram-negative pathogens including extended spectrum β-lactamase producing E. coli. All isolated peptides were observed to be proteinaceous in nature and highly heat stable. Peptides were purified or partially purified using solid phase extraction followed by RP-HPLC. The mass of the peptides was determined using ESI or MALDI-TOF mass spectrometry. Tandem MS fragmentation of peptide NI04 generated several sequence tags. Draft genome sequences of the B. pumilus J1 and K. pneumoniae A7 producer strains were obtained using the Illumina MiSeq platform. This allowed identification of the genes encoding peptide NI04, which was confirmed to be novel and was named pumicin NI04. Further characterisation of pumicin NI04 demonstrated it was non-toxic to keratinocytes, Vero cells and non-haemolytic up to at least 18x the minimum inhibitory concentration. The discovery revealed that pumicin NI04 belongs to the WXG-100 peptide superfamily, having homology with the mycobacterial and staphylococcal virulence factor EsxA. This represents the first report of antimicrobial activity in a WXG-100 peptide and has intriguing evolutionary implications. Although it was not possible to fully characterise peptides NI03 and NI05, when BAGEL 3 was used to mine the B. pumilus J1 genome, a promising putative bacteriocin candidate was identified that was homologous to Enterocin AS-45, which also confers anti Gram-negative activity and may be related to the activity observed for NI03, however more evidence is required. Investigations of the K. pneumoniae A7 bacteriocin on the other hand helped establish that the K. pneumoniae microcin E492 pathway was present and highly conserved in strain A7, and is likely to be responsible for the activity observed indicating that NI05 was not a novel peptide.

Antibiotics have been used widely for the treatment of bacterial infections for over half a century. However, the emergence of resistance to antibiotics has aroused public health concern, leading to the development of antimicrobial peptides (AMPs) as potential alternative therapeutic agents against bacterial infections. AMPs are naturally found in many species and have important roles in our innate immune defense systems. AMPs are usually cationic amphipathic peptides with membrane destabilizing property. They have a relatively broad spectrum of antimicrobial activity and pathogens are less likely to develop resistance against AMPs. The major challenge of using AMPs as therapeutic agents is their toxicity towards mammalian cells. The biological stability of AMPs to protease in human body is another concern. To address the latter problem, instead of the naturally occur L-enantiomers, Denantiomeric AMPs were introduced to enhance their stability. This study aimed to test the hypothesis that the D-enantiomeric AMPs are more resistant than the Lenantiomeric AMPs against proteolytic degradation. Three pairs of synthetic D-/LAMPs (D-LAO160-P13/LAO160-P12; D-LAO160-H/LAO160-H; and D-LAK-120-HP13/LAK-120-HP13) were employed to test for their stability when treated with trypsin, serum and gastric fluid, and the samples were analyzed by high performance liquid chromatography (HPLC). Generally, all the D-enantiomeric AMPs were found to be resistant towards proteolysis. Besides, to compare the cytotoxicity of D-/LAMPs, MTT and LDH assays of the D/L-LAK120-HP13 pair were carried out on two different cell lines, A549 cells (human lung adenocarcinoma epithelial cells) and RAW264.7 cells (mouse macrophage cells). Significant difference in cytotoxicity of D-LAK120-HP13 and LAK120-HP13 on RAW264.7 cells were obtained from MTT assay, but not in LDH assays or on A549 cells. Further analysis has to be done to validate the findings obtained from this research.
published_or_final_version
Pharmacology and Pharmacy
Master
Master of Medical Sciences

La multiplication des résistances aux antibiotiques constitue une grave menace qui nécessite de nouvelles stratégies antimicrobiennes. Le but de ce travail est d'étudier le potentiel des nanocapsules lipidiques (NCLs) pour l'administration de peptides antimicrobiens (PAMs). Les premiers travaux ont porté sur le développement et l'optimisation de NCLs chargées en PAMs. Différentes stratégies d’association ont été testé (adsorption à la surface ou encapsulation dans le coeur de NCLs). Les résultats ont démontré une efficacité d'association comprise entre 20 et 40%, lorsque les peptides sont adsorbés à la surface et plus de 80%,lorsqu’ils sont encapsulés. La deuxième partie s’est concentrée sur l'évaluation de l’activité des complexes PAMs et NCLs ainsi que leur stabilité vis-à-vis des protéases. Les résultats ont montré que l'adsorption entraîne une potentialisation de l'activité antimicrobienne des PAMs, associée à une protection partielle contre la dégradation enzymatique. Inversement, l’encapsulation des PAMs montre une meilleure stabilité aux enzymes, corrélée à une efficacité d'encapsulation supérieure sans amélioration de l'activité antimicrobienne in vitro. Dans une troisième partie, les mécanismes impliqués dans les interactions LNC/PAM ainsi que l'interaction du complexe avec un modèle de membrane bactérienne ont été étudiés. Il a été montré que la structure et la flexibilité des PAMs aux interfaces solide-liquide gouverneraient l'adsorption des peptides à la surface des NCLs, entrainant un changement de leur comportement avec les membranes bactériennes. L’ensemble de ces résultats démontre le potentiel prometteur des NCLs en tant que vecteur de PAMs
The rapid increase in drug-resistant infections presents an acute problem in the healthcare sector, generating interest in novel antimicrobial strategies. The aim of this work is to explore the potential of lipid nanocapsules (LNCs) for Antimicrobial peptides (AMPs) delivery. Firstly, the experiments were focused on the development and optimization of AMP-loaded LNCs. Different strategies were investigated to deliver AA230,LL37 and DPK060 using LNCs (peptides adsorption atthe surface or encapsulated in the core of modified LNCs). The results demonstrated an association efficiency of 20 to 40%, when peptide is adsorbed, and over 80% encapsulation efficiency, when peptides are encapsulated. The second part concerned the study ofthe influence of peptides loading on their activity and stability against proteases. The results showed that peptides adsorption induced a potentiation of the antimicrobial activity of the native peptides, with a partial protection against proteolytic degradation. Conversely, peptides encapsulation allowed better peptide stability, correlated with higher encapsulation efficiencies and a preservation of the in vitro antimicrobial activity. In a third part, the mechanisms involved in LNC/AMP interactions and the complex interaction with model bacterial membrane have been evaluated. It has shown that structure and flexibility at solid-liquid interfaces govern peptide adsorption on the surface of the LNCs, which in turn is expected to change LNCs properties and interaction with bacterial membranes. Taken together, these results demonstrate the potential of LNCto deliver AMPs as an alternative anti-infective therapy