Probiotics have been defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. The beneficial effects of probiotics in the gut are well described and roles including immunomodulation and colonisation resistance have been documented. Recent reports suggest that topical use of probiotic bacteria may be an effective strategy to promote skin health or inhibit disease. Therefore, in this thesis the potential of probiotics to protect skin from pathogenic bacteria was assessed using primary keratinocytes as a model system, and the skin pathogen, Staphylococcus aureus. The ability of three probiotics, L. reuteri ATCC 55730, L. rhamnosus AC413 and L. salivarius UCC118 to inhibit the growth of S. aureus was tested using well-diffusion assays and spot on the lawn assays. All three probiotics inhibited the growth of S. aureus in well-diffusion assays, though this property was dependent on growth medium. Inhibition of S. aureus growth was principally via the production of organic acids rather than bacteriocin production. Next, to determine whether probiotics could protect keratinocytes, confluent normal human epidermal keratinocytes (NHEK) were infected with S. aureus (106 CFU/ml) in the presence or absence of the probiotic (108 CFU/ml). NHEK viability was measured using trypan blue exclusion assays. L. reuteri had a significant protective effect on NHEK when applied 1h prior to (P=0.0003), or simultaneously with S. aureus (P=0.002). L. reuteri did not however protect NHEK when applied 1h after S. aureus addition. There was no change in the number of viable S. aureus in cell culture assays. To determine whether the protective effect was due to the inhibition of adhesion, NHEK were either pre-exposed to the probiotic for 1h, simultaneously exposed to the probiotic and S. aureus for 1h, or exposed to the probiotic 30 minutes after S. aureus addition for 1h. Pre-exposure of NHEK to L. reuteri (exclusion) and simultaneous exposure to L. reuteri and S. aureus (competition) resulted in significantly less staphylococci adhering to NHEK (P=0.03 and P=0.008 respectively). However when L. reuteri was added after S. aureus (displacement), the number of adherent staphylococci was not reduced. The necessity of S. aureus adherence for the inactivation of NHEK was demonstrated using a α5β1 integrin blocking antibody. Finally, to compare the innate response of NHEK to probiotics with S. capitis and S. aureus, TLR-2, antimicrobial peptide (AMP) expression and IL-8 production were measured. TLR-2 protein (but not mRNA) expression was reduced in the presence of S. aureus (P=0.018). NHEK pre-exposed to S. capitis prior to S. aureus infection however, exhibited elevated TLR-2 protein and mRNA expression (P<0.0001 and P=0.009 respectively). NHEK pre-exposed to L. reuteri prior to S. aureus had no significant change in TLR-2 expression compared to untreated controls. ELISAs demonstrated that IL-8 production was significantly increased in NHEK pre-exposed to L. reuteri prior to S. aureus infection (P=0.0001). In conclusion, L. reuteri protected NHEK from the toxic effects of S. aureus at least partly through competitive exclusion of binding sites on NHEK. Finally, NHEK innate responses to probiotic bacteria were akin to those to the skin commensal, S. capitis. L. reuteri induced expression of a neutrophil chemoattractant, suggesting it could be of importance in priming the innate immune response against S. aureus infections. Taken together, these results suggest that probiotic bacteria could be used prophylactically within skin creams and soaps to prevent S. aureus colonisation and infection in skin.

Les peptides antimicrobiens sont des éléments clés des mécanismes d'immunité innée développés pour combattre les microorganismes. Parmi ceux-ci, il y a les peptides antifongiques dont l'un, la mytimycine (MytM), avait été partiellement décrite chez la moule Mytilus edulis. Les buts de cette thèse consistaient en la caractérisation complète de la MytM chez M. edulis et chez M. galloprovincialis, ainsi qu'en la compréhension du rôle de ce peptide dans l'immunité de la moule. Les résultats montrent (i) une diversité des séquences nucléotides et en acides aminés en fonction de l'origine géographique des moules, résultant probablement d'une adaptation aux conditions environnementales; (ii) que 2 gènes différents codant la MytM sont simultanément présents dans le génome d'une même moule; (iii) que le niveau d'expression du gène de la MytM dépend de la nature du stimulus, suggérant l'existence de processus de reconnaissance spécifiques; et (iv) que le niveau d'expression du gène de la MytM varie d'une moule à l'autre. En conclusion, la MytM joue un rôle essentiel et spécifique chez la moule. Les données apportées par cette thèse enrichissent notre connaissance sur l'immunité innée des invertébrés. / Antimicrobial peptides are crucial elements of the innate immune mechanisms developed to fight microorganisms. Among them are antifungal peptides from which one, named mytimycin (MytM), had been partially reported in the blue mussel, Mytilus edulis. The purposes of this thesis were to fully characterize MytM in M. edulis and M. galloprovincialis and to understand how this peptide participates in mussel immunity. Results showed (i) the diversity of MytM mRNA and translated amino acid sequences according to geographic origin of mussels, probably resulting from adaptation to their environments; (ii) that 2 different MytM genes are simultaneously present in the genome of the same individual mussel; (iii) that expression level of MytM gene depends on the nature of the challenge, suggesting specific recognition processes; and (iv) MytM expression level was different from one mussel to another. In conclusion, MytM appeared to play a prominent and specific role in mussels. The advancement of our works added new data to the knowledge of innate immunity in invertebrates.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-12T14:29:17Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Tese_Gazzaneo (Biblioteca Central).pdf: 2832068 bytes, checksum: bb29fc38317d9f60a0d6129655b9b7a1 (MD5) / Made available in DSpace on 2017-07-12T14:29:17Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Tese_Gazzaneo (Biblioteca Central).pdf: 2832068 bytes, checksum: bb29fc38317d9f60a0d6129655b9b7a1 (MD5) Previous issue date: 2016-03-14 / Defensinas são peptídeos antimicrobianos (AMPs) que apresentam atividade contra diversos microrganismos patogênicos, em especial fungos. Embora não totalmente elucidados, há diversos mecanismos de ação propostos para as defensinas, que incluem permeabilização seletiva ou ruptura da membrana plasmática de microorganismos, ação direta em alvos intracelulares, ativação de cascatas de sinalização e aumento da produção de espécies reativas de oxigênio. Desde a sua descoberta e, tendo em vista sua ampla atividade biológica, o uso de defensinas no melhoramento de plantas cultivadas, bem como na produção de novos medicamentos tem sido proposto. Estudos de atividade biológica e possível aplicação biotecnológica das defensinas demandam uma grande quantidade dessas proteínas. Entretanto, o processo de extração da mesma é laborioso, dispendioso e, de acordo com a população ou disponibilidades da espécie vegetal escolhida, não sustentável ecologicamente. Portanto, a utilização de sistemas heterólogos de expressão é uma importante ferramenta para obtenção de defensinas recombinantes em escala industrial. Nesse estudo, um gene de defensina “DeHys”, isolado da Euphorbia hyssopifolia, foi inserido no plasmídeo pET102/D-TOPO e células da linhagem BL21(DE3) de Escherichia coli foram transformada com essa construção. Foi produzida a defensina recombinante Dehys com tamanho aproximado de 24 kDa. Sua identidade foi confirmada por western blot e pela análise do padrão de digestão com proteases. / Defensins are antimicrobial peptides (AMPs) , which present activity against a variety of pathogenic microorganism, especially funghi. Although not completely elucidated, there are a variety of proposed mechanisms of action for defensins, which includes selective microorganisms plasmatic membrane permeabilization or rupture, straight action agains intracellular targets, activation of signaling cascades and the burst of reactive oxygen species. Since it’s discovery and due to it’s wide biological activities, it’s use in crop enhancing, as well as its use in the development of new drugs have been proposed. Defensin’s biological activity and biotecnological application studies demand a reasonable amount of purified protein. However, the extraction processes is laborious, expensive, time consuming and depending on the population or the chosen plant species supply, not ecologically sustainable. So, the use of heterologous expression sytems is an importante tool to obtain purified proteins in industrial scale. In this study, a defensing gene (Dehys) isolated from Euphorbia hyssopifolia was inserted in a p pET102/D-TOPO vector and trasnformed into BL21(DE3) Escherichia coli strains. A recombinat Dehys defensin of approximately 24 kDa was obtained. It’s identity was double-checked using by Western blot and protease digestion pattern analyses.

O aumento na incidência de infecções fúngicas e a alta toxicidade ou elevado índice de resistência associado aos antimicóticos comerciais, criou um mercado carente de novas drogas. Neste contexto, os peptídeos antimicrobianos (AMPs) surgem como uma alternativa promissora ou fonte de conhecimento por desempenhar ação inibidora de crescimento e/ ou letal contra bactérias Gram-positivas e Gram-negativas, fungos, parasitas e/ ou vírus, além de atividade antitumoral e efeito imunomodulador. Como os mecanismos pelos quais eles o fazem são diferentes daqueles das drogas não peptídicas, os AMPs estão pouco associados ao desenvolvimento de resistência microbiana. A hemoglobina (Hb) é uma fonte de peptídeos com funções biológicas diversas. O fragmento 33-61 (Hb33-61) da cadeia &#945; da Hb bovina foi o primeiro AMP descrito a ser gerado in vivo no trato gastrointestinal do carrapato Boophilus microplus. Nossos estudos posteriores usando CD e H1-RMN revelaram que a amidação C-terminal deste fragmento o tornava ainda mais ativo que o primeiro e que em presença de micelas de SDS o Hb33-61a apresenta uma dobra &#946; na porção N-terminal (Lys40-Phe43) e outra (Ser49- Ser52) seguida de &#945;-hélice no C-terminal (Ala53-Ala60), bem como um segmento Pro44-Leu48 capaz de mover-se independentemente e agir como uma dobradiça. Nossas investigações usando análogos sintéticos truncados do Hb33-61a mostraram que o Hb40-61a poderia ser sua porção mínima ativa por apresentar comportamento conformacional idêntico. Nossos estudos subsequentes enfocando as suas propriedades evidenciaram a sua capacidade de causar morte rápida de cepas de Candida, incluindo C. albicans resistentes ao fluconazol e extravasamento de conteúdo e formação de poros em LUVs, revelando sua ação permeabilizante de membrana. Em continuidade ao estudo do Hb40-61a, investigamos no presente trabalho as suas propriedades e o seu mecanismo de ação contra C. albicans. Para isso, sintetizamos, purificamos e caracterizamos esta hemocidina, o seu análogo inteiro composto por D aminoácidos (ent-Hb40-61a) e o seu análogo marcado com 5 (6) carboxifluoresceína (FAM-Hb40-61a). Ensaios com eritrócitos humanos confirmaram a baixa atividade hemolítica desses AMPs em meio de alta e baixa força iônica. O análogo ent-Hb40-61a apresentou a mesma atividade antifúngica que o análogo L, evidenciando um mecanismo de ação não-estereoespecífico. Análises de células de Candida tratadas com FAM-Hb40-61a por microscopia confocal mostraram que em ½ MIC e MIC o peptídeo deposita-se na membrana plasmática e é internalizado, respectivamente. Citometria de fluxo demonstrou que na MIC cerca de 97% das células encontram-se marcadas pelo peptídeo, confirmou a influência negativa da alta força iônica em sua atividade, mostrou que a internalização celular na MIC é independente da temperatura e que a alteração no metabolismo energético da célula afeta de maneira negativa a internalização do peptídeo. Ensaios de permeabilidade celular com Syto 09 e iodeto de propídeo confirmaram danos progressivos à membrana plasmática de C. albicans com o aumento da concentração do Hb40-61a. Experimentos usando DiBAC4(5) e de DPH revelaram que o Hb40-61a altera o potencial de membrana e afeta sua fluidez, respectivamente. Imagens preliminares das células tratadas e não tratadas com Hb40-61a por microscopia de força atômica (AFM) sugeriram alterações nas células de C. albicans após tratamento com a hemocidina. Medidas preliminares do diâmetro médio das células de C. albicans revelaram que elas diminuem após o tratamento com o peptídeo, o que pode ser mais um indício de dano à membrana plasmática por formação de poros e extravasamento de conteúdo intracelular. Assim, obtivemos fortes indícios de que o alvo do Hb40-61a é, de fato, a membrana plasmática das células de Candida, de que ele apresenta potencial de uso tópico para tratamento de candidíase e pode servir como modelo para o desenho de novas drogas antimicrobianas, peptídicas ou não, com propriedades ainda mais valiosas e índices terapêuticos mais elevados. Testes preliminares mostraram que é possível a adsorção do Hb40-58a à nanopartículas de PSS e que, em relação ao peptídeo livre, este arranjo mantém a atividade antifúngica com MIC superior e apresenta menor atividade hemolítica. / The increased incidence of fungal infections and the high toxicity or high level of resistance associated with conventional antimycotics created a demand for new drugs. Antimicrobial peptides (AMPs) constitute a promising alternative and/or an important source of knowledge due to their growth inhibitory action and/or lethality against Gram-positive and Gram-negative bacteria, fungi, parasites and/or viruses. Besides, AMPs display antitumoral and immunomodulator effects. As their mechanisms of action are different from those of non-peptide drugs, AMPs are less associated with the development of antimicrobial resistance. Hemoglobin (Hb) is a source of peptides with diverse biological functions. The fragment 33-61 (Hb33-61) of bovine Hb &#945; chain was the first AMP reported to be generated in vivo in gastrointestinal tract of Boophilus microplus. Our studies of Hb33-61 using CD and H1-NMR showed that amidation of its C-terminal (Hb33-61a) increases its activity; in the presence of SDS micelles, Hb33-61a is characterized by a central hinge joining the C-terminal region (containing a &#946; turn followed by a helical element) to the N-terminal region (that presents only a &#946; turn). Our previous investigations using synthetic truncated analogues of Hb33-61a suggested that Hb40-61a could be its minimal active portion as it presented equal biological and structural properties. Our subsequent studies focusing on its properties showed its ability to quickly kill Candida albicans strains (including those resistant to fluconazole) and to cause leakage of the contents of LUVs and pore formation in GUVs, revealing its membrane permeabilizing action. We further investigated the properties of Hb40-61a and its possible mechanism of action against C. albicans. To do it, we synthesized, purified and chemically characterized it, its all-D analogue (ent-Hb40-61a) and its analogue labeled with 5 (6) carboxyfluorescein (FAM-Hb40-61a). Tests using human erythrocytes confirmed the low toxicity of these hemocidins at high or low ionic strength. The ent-analogue was as active as the all-L compound suggesting a non stereospecific mechanism of action. Confocal microscopy analysis of Candida cells treated with FAM-Hb40-61a showed that at ½ MIC and MIC, the peptide deposits on the plasma membrane and is internalized, respectively. Flow cytometry results showed that at the MIC about 97% of the cells are marked by the peptide, confirmed the negative influence of high ionic strength on its antifungal activity and showed that the cellular internalization at the MIC is partially dependent on ATP, but independent on the temperature. Cell permeabilization assays using Syto 09 and propidium iodide confirmed progressive damage of the membrane as a function of Hb40-61a concentration. Experiments employing DiBAC4 (5) and DPH revealed that the Hb40- 61a alters the membrane potential and affects its fluidity, respectively. Preliminary atomic force microscopy (AFM) images of C. albicans cells before and after treatment with Hb40-61a suggested morphological changes in the plasma membrane. Preliminary measurements of the average diameters of the fungal cells indicated size reduction after treatment with the Hb40-61a probably resulting from pore formation and leakage of cell contents. Thus, we obtained strong evidences that the target of this peptide is indeed the plasma membrane of Candida cells. Thus, this hemocidin have the potential to be used topically for treating candidiasis and/or serve as model for the design of new antimicrobial drugs, peptide or non-peptide, with even more valuable properties and improved therapeutic indexes. Preliminary tests confirmed the possibility of adsorbing Hb40-58a to nanoparticles of polystyrene sulfate (PSS) and that resulting assembly is still active and less hemolytic than the free peptide.

Em artrópodes o sistema imune inato contribui para a adaptação de animais como os escorpiões à diferentes ambientes. Esse sistema é composto por mecanismos capazes de agir contra injúrias e a ação de microrganismos e entre esses mecanismos estão os peptídeos antimicrobianos (PAMs). O objetivo deste trabalho foi identificar PAMs presentes na hemolinfa de Tityus serrulatus. Para isso sua hemolinfa foi extraída e separados os hemócitos e plasma, em seguida fracionamos em 3 concentrações de acetonitrila em TFA 0,05% (05, 40 e 80%). Estas frações foram submetidas a uma cromatografia liquida de alta eficiência (CLAE) e os picos foram avaliados quanto a sua ação antimicrobiana e hemolítica. Foram identificadas 16 frações que apresentam atividade antimicrobiana. Uma das frações com atividade antimicrobiana, presente nos hemócitos apresentou similaridade com defensina descrita em carrapatos da espécie Ixodes scapularis. Essa fração possui aproximadamente 3486 Da, não apresenta atividade hemolítica e foi denominada como Serrulina. / In arthropods, its innate immune system contributes to the adaptation of animals like scorpions to different environments. This system consists of mechanisms that act avoiding injuries and against the action of microorganisms, among these mechanisms are antimicrobial peptides (AMPs). The aim of this study was to identify AMPs, present in the hemolymph of Tityus serrulatus. The hemolymph was extracted and then we separated hemocyte and plasma. The samples were fractionated in different concentrations of acetonitrile in TFA 0.05% (05, 40 and 80%). These fractions were subjected to high-performance liquid chromatography (HPLC) and the peaks obtained were evaluated for its antimicrobial and hemolytic action. We found sixteen fractions with antimicrobial activity. One of the fractions with antimicrobial activity, present in hemocytes, is similar with a defensin described in ticks, Ixodes scapularis. This fraction has about 3486 Da, has no hemolytic activity and was named as Serrulina.

Os peptídeos antimicrobianos tem sido alvo de cada vez mais estudos como candidatos a antibióticos naturais devido a seu amplo espectro de ação e baixa suceptibilidade a induzir resistência microbiana. Carentes de sistema imune específico, os invertebrados contam com um eficiente sistema imune inato para se defenderem de microrganismos patogênicos, incluindo uma coleção de peptídeos antimicrobianos. Através de cromatografia, ensaios antimicrobianos, espectrometria de massas e análise em banco de dados, isolou-se frações do muco de lesmas da espécie P boraceiensis com efeito antimicrobiano e obteve-se sequencias mais prováveis de fragmentos de algumas das frações ativas encontradas, relacionando-os as proteínas e peptídeos conhecidos mais similares e sugerindo relação entre as sequencias encontradas, as proteínas e peptídeos similares, bem como possível relação com atividade antimicrobiana. Obteve-se uma gama de frações ativas contra bactérias e/ou leveduras e sequências e informações relacionadas que podem ser úteis para futuros estudos de isolamento e caracterização de fatores antimicrobianos no muco de P boraceiensis. / Antimicrobial peptides are becoming increasingly more important as natural antibiotics candidates since they show a broad spectrum of action and low suceptibility to induce microbial resistance. Lacking specific imunne system, invertebrates count with an efficient innate imunne system to deffend theirselves from pathogenic microrganisms, including a collection of antimicrobial peptides. Through chromatografy, antimicrobial tests, mass spectometry and database analysis, we isolated fractions from the mucucs of P boraceiensis slugs showing antimicrobial effect. We obtained most propable sequences from several active fractions, relating them with most similar known peptides and proteins. There were sugesteg some possible connections between the found sequences and related peptides and proteins as well as possible antimicrobial activity relations. We found several active fractions against bacteria and/or yeast as well as sequences and related information that may be usefull in further isolation and caracterization studies of antimicrobial factors in P boraceiensis mucus.

Antimicrobial peptides are among the repertoire of host innate immune defenses. In mucosal immunity, the health-disease balance can be greatly modulated by the interplay between host immune factors and colonized microflora. Microbial ecology within dental plaque is constantly shaped by environmental factors present within the oral cavity. Several antimicrobial peptides are detected in saliva and their bactericidal activities against oral bacteria, including Streptococcus mutans, the primary etiologic agent of dental caries, have been clearly demonstrated. However, the role of these antimicrobial peptides in S .mutans ecology and host caries experience is not well-defined. We hypothesized that various strains of S. mutans possess different inherent susceptibility/resistance profiles to host salivary antimicrobial peptides and that host-specific quantities of these peptides may influence plaque colonization by particular S. mutans strains. S. mutans strains from subjects with variable caries experience were tested for susceptibility to a panel of antimicrobial peptides, including HNP-1-3, HBD-2-3 and LL-37, revealing that the susceptibilities of S. mutans to these peptides were strain-specific. S. mutans strains from high caries subjects showed greater resistance to these peptides at varying concentrations than those from caries-free subjects. In addition, when combinations of these peptides were tested, they showed either additive or synergistic interaction against S. mutans. Determinations of the salivary levels of these peptides showed that their concentrations were highly variable among subjects with no correlation to host caries experience. However, positive relationships between the salivary concentrations of HNP-1-3 and MS in dental plaque were found. Additionally, the levels of a number of these peptides in saliva appeared to be positively correlated within an individual. An analysis of the salivary peptide concentrations and the susceptibility profiles of S. mutans strains showed that S. mutans strains obtained from subjects with higher concentration of HNP-1-3 in saliva appeared to be more resistant to HNP-1. Collectively, our findings showed that salivary antimicrobial peptides affect S. mutans ecology by restricting the overall growth of this bacterium within the oral cavity and that their activity may help select resistant strains of S. mutans to colonize within dental plaque. The relative ability of S. mutans to resist host salivary antimicrobial peptides may be considered a potential virulence factor for this species.

Orientador: Ary Fernandes Júnior / Resumo: O aumento no número de bactérias multirresistentes aos fármacos antibacterianos é preocupação de saúde pública e tem motivado pesquisas na buscsa por antimicrobianos alternativos para minimizar este problema, e na obtenção de substâncias com capacidade de matar bactérias e/ou interferir com a sua patogenicidade. O Staphylococcus aureus é uma bactéria altamente virulenta, capaz de causar inúmeras doenças, incluindo intoxicações alimentares. Esta bactéria se tornou resistente aos diversos antimicrobianos ao longo dos anos com destaque para o S. aureus meticilina-resistente (MRSA). O peptídeo antimicrobiano (AMP) nisina, bacteriocina produzida por Lactococcus lactis é um que vem sendo estudado na forma de nanoemulsões e nanopartículas. O objetivo desse estudo foi sintetizar, caracterizar e testar nanoemulsões e nanopartículas de ouro (AuNPs) de nisina, para a verificação da ação antibacteriana, através da Concentração Inibitória Mínima (CIM), atividade antibiofilme, antienterotoxina, atividade hemolítica, ação sobre a membrana bacteriana determinada pelo extravasamento de proteínas, sobre linhagens padrões ATCC de S. aureus, e teste de viabilidade celular em linhagem HCT-116 por citometria de fluxo. Os tratamentos utilizados foram nisina, cinco nanoemulsões com nisina (Nano-Nis), AuNPs com nisina (AuNPs-Nis), AuNPs com Nano-Nis (AuNPs + Nano-Nis) e AuNPs-Nis com nanoemulsão (AuNPs-Nis + Nano). De acordo com os resultados obtidos para CIM, observou-se que para a cepa ATCC 33591 d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The growth in the number of multiresistant bacteria resistant to traditional antimicrobial drugs is a public health concern which has been motivated researchs worldwide, seeking new antimicrobial drugs to minimize this problem besides getting new substances able to erradicate bacteria and/or interfer with their pathogenicity. Staphylococcus aureus is a highly virulent bacteria, able to cause countless diseases including food poisoning. This bacteria got resistant to many antimicrobials within the years, highlighting the S. aureus metchilin-resistant (MRSA). The antimicrobial peptide (AMP) nisin, bacteriocin synthesized by Lactococcus lactis, is one that has been studied in nanoemulsions and nanoparticles. The objective of this study was to synthesize, characterize and evaluate nanoemulsions and gold nanoparticles (AuNPs) with nisin, to verify their antibacterial action, using the Minimum Inhibitory Concentration (MIC), antibiofilm activity, antienterotoxin activity, hemolytic activity, action on bacterial membrane determined by protein leakage, on MRSA ATCC strains, and cell viability in HCT-116 strain by flow citometry. The treatments used were nisin, five nanoemulsions with nisin (Nano-Nis), AuNPs with nisin (AuNPs-Nis), AuNPs with Nano-Nis (AuNPs + Nano-Nis) and AuNPs-Nis with nanoemulsion (AuNPs-Nis + Nano). According to the results obtained for MIC, it was observed that for the MRSA strain ATCC 33591, the number 1 formulation of nanoemulsions was the more efficient betwe... (Complete abstract click electronic access below) / Mestre

Healthcare associated infections (HAIs) cost the US healthcare system over $45 billion to treat and cause millions of deaths annually. A large subset of HAIs are associated with medical devices that are meant to improve and save lives. Infected devices are treated using traditional antibiotics, contributing to development of antibiotic resistance. Antibiotic resistance is expected to cost $100 trillion and kill more people a year than cancer by 2050; thus, new alternative antimicrobials for the treatment of device-associated HAIs are critically needed. Antimicrobial peptides (AMPs), such as 26 amino-acid (aa), marine-derived Chrysophsin-1 (CHY1), are poised to reduce HAIs due to their broad antimicrobial activity and unique mechanisms of action that do not promote bacterial resistance. AMPs are short (12-50aa), positively charged (+2-+9) proteins found in the innate immune systems of many different species. Their high separation of hydrophilic and hydrophobic residues leads to many unique mechanisms derived from many unique secondary and tertiary structures that are not yet well understood. Despite the discovery of over 2000 natural AMPs and many more synthetically designed AMPs, none have been successfully commercialized for healthcare applications due to challenges surrounding cytotoxicity, short in vivo half-life (degradation), high costs of production and effectiveness in physiological environments (such as those with high-salt). Several strategies have been investigated to overcome these challenges, for example, truncation of cytotoxic sequences or D-amino acid substitution to improve AMP toxicity and stability; however, many of these strategies can reduce antimicrobial effectiveness. A unique strategy of increasing stability, reducing cytotoxicity, and maintaining antimicrobial activity that is relevant for medical devices is the covalent tethering (binding) of AMPs via a flexible tethering molecule to the surface. However, the effect of tethering parameters on resulting AMP mechanisms and activity is still widely debated. AMP activity can vary widely by utilizing different tethering strategies, which include additional variables such as: (1) peptide choice and properties (such as native mechanism, concentration, charge, and structure), (2) tether choice and properties (such as chemical composition, length, charge, surface density, and flexibility), and (3) testing conditions (such as temperature, solvent composition and substrate type). Some studies suggest that AMP performance may be tether-dependent, for example some AMPs require longer tethers while others do not and some need a flexible tether. Thus, models for predicting successful tethering strategies for different AMP properties, which currently do not exist, must be developed. Further, complicated and often destructive techniques, such as XPS and SEM, are typically implemented to study the relationship of all these parameters vs. antimicrobial activity, which are labor-intensive and limited in scope. Predictive models guiding tether strategy need to be constructed, but also new techniques to study tethering be developed. If these technical milestones are achieved they can serve as a predicate for commercial implementation of a host of new therapies targeted at reducing device-associated HAIs. The overall goal of this thesis was to study the relationship between antimicrobial activity of tethered C-CHY1 examining both spacer length and peptide surface density and the development of a feasible clinical business case for tethered AMPs. To achieve this goal, a traditional entrepreneurial approach was taken in which a minimally-viable product was first designed and business case analyzed, followed by studies to better optimize and understand the underlying structure-mechanism relationships. CHY1 with a C-terminus cysteine to allow for surface-binding (C-CHY1) was tethered onto a silicon dioxide surface via a flexible poly(ethylene glycol) (PEG) tether, and then both surface binding behavior and antimicrobial success of C-CHY1 were examined as a function of tether properties and reaction conditions. For these studies, quartz-crystal microbalance with dissipation (QCM-D) was the primary technique, a real-time, non-destructive flow method that was then coupled with downstream characterization techniques: fluorescent microscopy and contact angle measurements. In parallel a deep dive into domestic and international business models for commercializing AMP technologies. Specifically, tether length and surface density effects on C-CHY1 mechanisms were studied, followed by the effect of temperature, type of microbe, and salt concentration on the antimicrobial mechanisms of tethered C-CHY1. QCM-D was used to measure binding of C-CHY1 via three different length tethers, PEG molecular weight (MW) 866, 2000 and 7500, followed by microscopy to measure antimicrobial effectiveness against two model microbes Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Modeling of QCM-D data allowed for surface density and thickness to be calculated and related to C-CHY1 antimicrobial activity. PEG 7500 allowed proper C-CHY1 orientation and mobility, allowing for its native pore-forming mechanism and highest activity while PEG 866 tethers led to denser grafting and an effective, yet non-native ion displacement mechanism. The QCM-D was used to characterize the effect of salt concentration and temperature reaction conditions on the grafting density of C-CHY1 tethered via PEG 866 and PEG 7500, which was then related to antimicrobial activity. For PEG MW 866, neither temperature nor salt concentration increases significantly altered the grafting density of C-CHY1 while for PEG 7500 increasing temperature allowed for significantly increased grafting density. C-CHY1 density had no significant effect on antimicrobial activity against either microbe. Temperature of bacterial incubation did demonstrate microbe-specific changes in C-CHY1 antimicrobial activity. These results demonstrated that small changes in reaction conditions can drastically change membrane selectivity of C-CHY1. An in-depth investigation of the effects of bacterial membrane composition and temperature on soluble C-CHY1 mechanism was implemented to better understand the molecular membrane- and temperature-dependent selectivity and structure-function of C-CHY1. Supported lipid bilayers (SLBs) formed in QCM-D can be used as model membranes to elucidate AMP action mechanisms against membranes of different compositions. Two and three component SLBs representative of Gram-negative phosphatidylethanolamine (PE) and phosphatidyglycerol acid (PG) with and without charged lipopolysaccharide, LPS and Gram-positive bacteria phosphatidylcholine (PC) and PG with and without charged lipoteichoic acid, (LTA) were formed at both 23°C and 37°C. C-CHY1 at 5 µM was exposed to the different membranes and mechanistic surface action was studied. The membranes formed highly different baseline responses in QCM-D, indicative of vastly different membrane structures, thicknesses and deposition behaviors on SiO2, warranting future studies. Further, significant effects of LTA incorporation were observed in both peptide interaction and deposition. There were measurable effects of temperature on membrane formation as well as peptide interaction kinetics and even mode of interaction. Lastly, business models for the commercialization of novel medical device technologies such as surface-tethered C-CHY1 were investigated. While this technology has the potential to solve many unmet needs, there must a commercialization plan implemented in order to have an impact. There is a clear disconnect between technology development in academia and technology commercialization in industry that must be connected. Development of an entrepreneurial mindset at the graduate school level, can help bridge the gap. A thorough investigation of domestic and international business models for commercializing AMP technologies was carried out and distilled in the form of the Business Model Canvas developed by Alexander Osterwalder that can be used as a roadmap for commercialization efforts. Using the QCM-D a relationship between both spacer length and peptide surface density and the antimicrobial activity of tethered C-CHY1 was determined. A business plan was developed in order to increase the impact of this and other AMP based work. This work provides a roadmap for future researchers to quickly develop and commercial novel AMP based coating technology.

Healthcare associated infections (HAIs) cost the US healthcare system over $45 billion to treat and cause millions of deaths annually. A large subset of HAIs are associated with medical devices that are meant to improve and save lives. Infected devices are treated using traditional antibiotics, contributing to development of antibiotic resistance. Antibiotic resistance is expected to cost $100 trillion and kill more people a year than cancer by 2050; thus, new alternative antimicrobials for the treatment of device-associated HAIs are critically needed. Antimicrobial peptides (AMPs), such as 26 amino-acid (aa), marine-derived Chrysophsin-1 (CHY1), are poised to reduce HAIs due to their broad antimicrobial activity and unique mechanisms of action that do not promote bacterial resistance. AMPs are short (12-50aa), positively charged (+2-+9) proteins found in the innate immune systems of many different species. Their high separation of hydrophilic and hydrophobic residues leads to many unique mechanisms derived from many unique secondary and tertiary structures that are not yet well understood. Despite the discovery of over 2000 natural AMPs and many more synthetically designed AMPs, none have been successfully commercialized for healthcare applications due to challenges surrounding cytotoxicity, short in vivo half-life (degradation), high costs of production and effectiveness in physiological environments (such as those with high-salt). Several strategies have been investigated to overcome these challenges, for example, truncation of cytotoxic sequences or D-amino acid substitution to improve AMP toxicity and stability; however, many of these strategies can reduce antimicrobial effectiveness. A unique strategy of increasing stability, reducing cytotoxicity, and maintaining antimicrobial activity that is relevant for medical devices is the covalent tethering (binding) of AMPs via a flexible tethering molecule to the surface. However, the effect of tethering parameters on resulting AMP mechanisms and activity is still widely debated. AMP activity can vary widely by utilizing different tethering strategies, which include additional variables such as: (1) peptide choice and properties (such as native mechanism, concentration, charge, and structure), (2) tether choice and properties (such as chemical composition, length, charge, surface density, and flexibility), and (3) testing conditions (such as temperature, solvent composition and substrate type). Some studies suggest that AMP performance may be tether-dependent, for example some AMPs require longer tethers while others do not and some need a flexible tether. Thus, models for predicting successful tethering strategies for different AMP properties, which currently do not exist, must be developed. Further, complicated and often destructive techniques, such as XPS and SEM, are typically implemented to study the relationship of all these parameters vs. antimicrobial activity, which are labor-intensive and limited in scope. Predictive models guiding tether strategy need to be constructed, but also new techniques to study tethering be developed. If these technical milestones are achieved they can serve as a predicate for commercial implementation of a host of new therapies targeted at reducing device-associated HAIs. The overall goal of this thesis was to study the relationship between antimicrobial activity of tethered C-CHY1 examining both spacer length and peptide surface density and the development of a feasible clinical business case for tethered AMPs. To achieve this goal, a traditional entrepreneurial approach was taken in which a minimally-viable product was first designed and business case analyzed, followed by studies to better optimize and understand the underlying structure-mechanism relationships. CHY1 with a C-terminus cysteine to allow for surface-binding (C-CHY1) was tethered onto a silicon dioxide surface via a flexible poly(ethylene glycol) (PEG) tether, and then both surface binding behavior and antimicrobial success of C-CHY1 were examined as a function of tether properties and reaction conditions. For these studies, quartz-crystal microbalance with dissipation (QCM-D) was the primary technique, a real-time, non-destructive flow method that was then coupled with downstream characterization techniques: fluorescent microscopy and contact angle measurements. In parallel a deep dive into domestic and international business models for commercializing AMP technologies. Specifically, tether length and surface density effects on C-CHY1 mechanisms were studied, followed by the effect of temperature, type of microbe, and salt concentration on the antimicrobial mechanisms of tethered C-CHY1. QCM-D was used to measure binding of C-CHY1 via three different length tethers, PEG molecular weight (MW) 866, 2000 and 7500, followed by microscopy to measure antimicrobial effectiveness against two model microbes Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Modeling of QCM-D data allowed for surface density and thickness to be calculated and related to C-CHY1 antimicrobial activity. PEG 7500 allowed proper C-CHY1 orientation and mobility, allowing for its native pore-forming mechanism and highest activity while PEG 866 tethers led to denser grafting and an effective, yet non-native ion displacement mechanism. The QCM-D was used to characterize the effect of salt concentration and temperature reaction conditions on the grafting density of C-CHY1 tethered via PEG 866 and PEG 7500, which was then related to antimicrobial activity. For PEG MW 866, neither temperature nor salt concentration increases significantly altered the grafting density of C-CHY1 while for PEG 7500 increasing temperature allowed for significantly increased grafting density. C-CHY1 density had no significant effect on antimicrobial activity against either microbe. Temperature of bacterial incubation did demonstrate microbe-specific changes in C-CHY1 antimicrobial activity. These results demonstrated that small changes in reaction conditions can drastically change membrane selectivity of C-CHY1. An in-depth investigation of the effects of bacterial membrane composition and temperature on soluble C-CHY1 mechanism was implemented to better understand the molecular membrane- and temperature-dependent selectivity and structure-function of C-CHY1. Supported lipid bilayers (SLBs) formed in QCM-D can be used as model membranes to elucidate AMP action mechanisms against membranes of different compositions. Two and three component SLBs representative of Gram-negative phosphatidylethanolamine (PE) and phosphatidyglycerol acid (PG) with and without charged lipopolysaccharide, LPS and Gram-positive bacteria phosphatidylcholine (PC) and PG with and without charged lipoteichoic acid, (LTA) were formed at both 23°C and 37°C. C-CHY1 at 5 µM was exposed to the different membranes and mechanistic surface action was studied. The membranes formed highly different baseline responses in QCM-D, indicative of vastly different membrane structures, thicknesses and deposition behaviors on SiO2, warranting future studies. Further, significant effects of LTA incorporation were observed in both peptide interaction and deposition. There were measurable effects of temperature on membrane formation as well as peptide interaction kinetics and even mode of interaction. Lastly, business models for the commercialization of novel medical device technologies such as surface-tethered C-CHY1 were investigated. While this technology has the potential to solve many unmet needs, there must a commercialization plan implemented in order to have an impact. There is a clear disconnect between technology development in academia and technology commercialization in industry that must be connected. Development of an entrepreneurial mindset at the graduate school level, can help bridge the gap. A thorough investigation of domestic and international business models for commercializing AMP technologies was carried out and distilled in the form of the Business Model Canvas developed by Alexander Osterwalder that can be used as a roadmap for commercialization efforts. Using the QCM-D a relationship between both spacer length and peptide surface density and the antimicrobial activity of tethered C-CHY1 was determined. A business plan was developed in order to increase the impact of this and other AMP based work. This work provides a roadmap for future researchers to quickly develop and commercial novel AMP based coating technology.