Tritrpticina (TRP3) é um peptídeo antimicrobiano com 13 resíduos de amino ácidos com três Ws sequenciais. Com o objetivo de contribuir para a compreensão de seu mecanismo de ação, realizaram-se estudos funcionais e conformacionais da TRP3 e de dois análogos onde um (WLW) ou dois (LWL) W foram substituídos por L. Os peptídeos foram igualmente ativos contra bactérias Gram positivas e negativas. Sua atividade hemolítica requereu concentrações maiores, diminuindo na ordem TRP3>WLW>LWL. Os peptídeos permeabilizaram membranas modelo de E. coli ou contendo fosfolipídios carregados negativamente. Espectros de CD sugeriram que os peptídeos adquirem diferentes conformações ao se ligarem a bicamadas e micelas. Estudos de fluorescência mostraram que a ligação a membranas decresce na ordem: TRP3>WLW>LWL e que os peptídeos se localizam próximos à interface membrana-água. Espectros de RPE de marcadores de spin lipídicos indicaram que a ligação dos peptídeos altera a organização dos lipídios, aumentando o empacotamento molecular / Tritrpticin (TRP3) is a 13-residue antimicrobial peptide that contains three sequential Ws. With the aim of contributing to the understanding of its mechanism of action, functional and conformational studies were performed with TRP3 and two of its analogues where one (WLW) or two (LWL) of the W were replaced by L. The peptides were equally active against both Gram positive and Gram negative bacteria. Higher concentrations were required for hemolytic activity which varied in the order: TRP3>WLW>LWL. The peptides permeabilized membranes model membranes mimicking E. coli\'s lipid composition or containing different negatively charged phospholipids. CD spectra suggested the peptides acquired different conformations upon binding to bilayers or micelles. Fluorescence studies showed that membrane binding decreases in the order: TRP3>WLW>LWL and that the peptides are located close to the water-membrane interface. EPR spectra of lipid spin labels indicated that peptide binding alter lipid organization, increasing molecular packing

Neste trabalho, estudamos a interação de um peptídeo antimicrobiano com membranas modelo, por meio de dicroísmo circular (CD), fluorescência e microscopia óptica. Tal peptídeo, chamado de híbrido, foi sintetizado como uma mistura das regiões mais ativas de dois outros peptídeos antimicrobianos, chamados de pediocina A e plantaricina 149. Esse peptídeo híbrido possui carga de +8, em pH fisiológico, e as membranas estudadas foram compostas por uma mistura de fosfolipídeos zwiteriônicos (cabeça polar de fosfatidilcolina, PC) e aniônicos (cabeça polar de fosfatidilglicerol, PG), em diferentes razões molares. Os resultados de CD evidenciaram que este peptídeo se apresenta de forma desordenada em solução aquosa, porém adota uma conformação helicoidal na presença de grandes vesículas unilamelares carregadas negativamente (LUVs). A quantidade de componente helicoidal é dependente da quantidade de lipídeo negativo presente na bicamada lipídica. A fluorescência do triptofano revelou um deslocamento para o azul muito significativo, chegando a 20 nm para membranas compostas por 100 mol% de PG. Os dois resultados (CD e fluorescência) indicam que a região dos aminoácidos que contém o triptofano deve estar interagindo muito fortemente com a região hidrofóbica da membrana, numa conformação tipo-helicoidal. Experimentos de vazamento de carboxifluoresceína encapsulada em LUVs, por espectroscopia de fluorescência, demonstraram a ação lítica do peptídeo induzindo a formação de poros nas membranas, independentemente da composição das LUVs. Entretanto, a razão molar peptídeo:lipídeo necessária para induzir vazamento da sonda foi menor para membranas lipídicas compostas por bicamadas contendo altas quantidades de PG. Tal fato coloca em evidência o papel fundamental da interação eletrostática entre os peptídeos carregados positivamente com as membranas carregadas negativamente para o processo de ligação e mecanismo de ação deste peptídeo. Para estudar mais detalhadamente o mecanismo de ação, realizamos experimentos de microscopia óptica em vesículas unilamelares gigantes. Concluímos que o peptídeo provoca total desestabilização das vesículas unilamelares gigantes, com formação de poros, seguidos de ruptura da bicamada lipídica e sua transformação em pequenos e mal definidos complexos de peptídeos e fosfolipídeos. / In this work, we investigated the interaction between an antibacterial peptide with model membranes, by means of circular dichroism (CD), fluorescence and optical microscopy. Such a peptide was synthesized from the most active regions of two others antimicrobial peptides, namely pediocin A and plantaricin 149. The hybrid peptide has a net charge of ~ +8, at physiological pH, and the studied model membranes were composed of a mixture of zwitterionic phospholipids (phosphatidylcholine polar head) and anionic phospholipids (phosphatidylglycerol polar head), at differente molar ratio. The CD results evidenced that the peptide was essentially structureless in aqueous solution, but acquired an helical conformation in the presence of charged large unillamellar vesicles LUVs. The helical content is dependent on the negative charge amount on membrane surface. The tryptophan fluorescence revealed a significant blue shift of the maximium emission wavelength, up to 20 nm for the membranes composed of 100 mol% of PG in respect to the peptide fluorescence in the aqueous solution. This indicates that part of the aminoacid residues, that contains the tryptophan, must be buried into the hydrophobic medium of the lipid membrane. Leakage experiments using fluorescence spectroscopy of carboxyfluorescein encapsulated in LUVs demonstrated the lytic action of the peptide, inducing the pore formation in the membrane, regardless of lipid membrane composition. However, it should be stressed that the peptide:lipid molar ratio necessary to induce probe leakage was smaller for lipid membranes made up of large PG amounts. Such evidence points out the key role of the electrostatic interaction between a positively charged peptide and the negatively charged membrane, mediated by hydrophobic contribution. To gain further insight into the lytic mechanism of the peptide, we performed single vesicle experiments using giant unilamellar vesicles under optical microscopy observations. We conclude that the peptide provokes a total membrane desestabilization, with pore formation, followed by a membrane disruption and its transformation into smaller and not well defined complexes of phospholipids and peptides.

A resistência aos antibióticos adquirida por micro-organismos patogênicos é um problema de saúde mundial e, por isso, o desenvolvimento de novos agentes antimicrobianos vem sendo amplamente estimulado. Sabendo que muitos peptídeos bioativos correspondem a fragmentos peptídicos de proteínas e/ou seus análogos, este trabalho teve o objetivo de desenvolver novos peptídeos antimicrobianos (AMPs) a partir das sequências aminoacídicas e das estruturas 3D de proteínas possivelmente envolvidas na atividade antimicrobiana de venenos de serpentes pouco estudados. As etapas iniciais seguidas foram: a) escolher uma fosfolipase A2 (PLA2) de veneno de serpente peruana do gênero Bothrops da família Viperidae com sequência de aminoácidos conhecida e modelar por homologia a sua estrutura 3D; b) verificar atividade antimicrobiana em venenos de serpentes peruanas dos gêneros Bothrops e Bothriopsis da família Viperidae, selecionar um veneno ativo, fracioná-lo para isolar proteínas provavelmente envolvidas nessa atividade, tripsinizar as proteínas isoladas, sequenciar os fragmentos trípticos para identificá-las, localizar esses fragmentos em sequências aminoacídicas de proteínas com estruturas 3D disponíveis correlatas às proteínas isoladas/identificadas em classe, função e fonte natural. Em seguida, foram escolhidos fragmentos peptídicos da PLA2 (item a) e das proteínas isoladas do veneno ativo (item b) e/ou desenhados análogos que apresentassem características exibidas por AMPs conhecidos. Os peptídeos desenhados foram sintetizados, purificados, caracterizados e testados em suas atividades antimicrobianas. Os modelos estruturais 3D da PLA2 de Bothrops pictus e quatro peptídeos (PLA2-1 a -4) amidados derivados dela foram obtidos, sendo o PLA2-1 ativo frente a Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Candida krusei e Candida parapsilosis (MICs de 6,25-200 µmol.mL-1). Dos três venenos de serpentes peruanas testados, Bothrops taeniatta, Bothrops barnetti e Bothriopsis oligolepis, os dois últimos inibiram o crescimento de S. aureus (MICs 0,78-50 µmol.mL-1), mas apenas B. oligolepis demonstrou espectro de ação amplo. O seu fracionamento sequencial, acompanhado de ensaios de inibição do crescimento de S. aureus, gerou frações ativas relativamente homogêneas que, tripsinizadas e os fragmentos trípticos sequenciados, continham metalo-peptidases do tipo III, serino-peptidase ou lectinas do tipo C. A verificação de atividade enzimática e de coagulação sanguínea nessas frações confirmaram as naturezas das proteínas isoladas. Dos três peptídeos amidados (Bo-Ser1, Bo-Met1 e Bo-Lec1) desenhados a partir de suas estruturas, um deles foi ativo frente às leveduras C. albicans, C. krusei e C. parapsilosis (Bo-Met1; MIC de 6,25 - 200 µmol.mL-1). Pela primeira vez, foi demonstrado que: a) os venenos das serpentes peruanas B. barnetti e B. oligolepis apresentam ação antimicrobiana, sendo o último de espectro amplo; b) que as proteínas acima citadas, que incluem uma serino-peptidase, estão envolvidas com essa propriedade do veneno de B. oligolepis; c) que as sequências aminoacídicas e modelo 3D de uma PLA2 ácida e de proteínas presentes nos venenos das serpentes peruanas B. pictus e Bothriopsis oligolepis podem funcionar como fontes naturais para o desenvolvimento de novos AMPs de ação potente em micro-organismos de interesse clínico e científico. / Resistance to antibiotics obtained by pathogenic microorganisms is a global health problem, so the search for new antimicrobial agents has been encouraged. Knowing that many protein fragments and analogues are bioactive peptides, the aim of this work was to develop new antimicrobial peptides (AMPs) based on the amino acid sequences and 3D structures of proteins apparently involved in the antimicrobial activity of snake venoms very little or not studied so far. The first steps taken were: a) selection of a phospholipase A2 (PLA2) present in the venom from a Peruvian Bothrops sp. belonging to the family Viperidae, whose amino acid sequence was known, to model by homology its 3D structure; b) detection of antimicrobial activity in venoms from other Peruvian Viperidae Bothrops and Bothriopsis snakes, selection of an active venom, fractionation of it for isolation of proteins possibly involved in the antimicrobial activity, trypsinization of the isolated proteins, sequencing of the tryptic fragments for protein identification, location of such fragments in the amino acid sequences and 3D structures of proteins directly related in class, function and natural source to the isolated proteins. Then, peptide fragments from the chosen PLA2 (item a) and from the isolated proteins (item b) that presented structural features found in the known AMPs were selected and/or their analogues were designed. Finally, synthesis, purification and characterization of the peptides with AMP potential, (viii) verification on whether or not they display antimicrobial activity. The 3D-structure models of Bothrops pictus PLA2 and four amidated peptides (PLA2-1 to -4) derived from it were obtained, being PLA2-1 active against Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa as well as the yeasts Candida albicans, Candida krusei and Candida parapsilosis (MICs de 6.25-200 µmol.mL-1). Among the three Peruvian snake venoms tested Bothrops taeniatta, Bothrops barnetti and Bothriopsis oligolepis, the last two inhibited the growth of S. aureus (MICs 0.78-50 µmol.mL-1) and B. oligolepis presented a wide spectrum of bacterial action. Sequential fractionation followed by S. aureus growth inhibition assays of the main fractions led to active relatively homogeneous ones. Their trypsinization and sequencing of the tryptic fragments indicated that they contained metalloproteinases type III, serine-proteinase or lectins type CTL. Enzymatic activity and blood coagulation assays confirmed the nature of the isolated proteins. From the three amidated peptides (Bo-Ser1, Bo-Met1 e Bo-Lec1) derived from them, Bo-Met1 showed to be active against C. albicans, C. krusei e C. parapsilosis (MIC 6,25 - 200 µmol.mL-1). In summary, for the first time, it was demonstrated that: a) the venoms of the Peruvian snakes B. barnetti and B. oligolepis display antimicrobial activity, being the last of wide spectrum of action, b) the proteins isolated from B. oligolepis snake venom, including a serine-peptidase, are involved in the antimicrobial activity of the B. oligolepis snake venom, c) the amino acid sequences and 3D structures of acidic PLA2 and of other proteins found in the venoms of the Peruvian B. pictus e Bothriopsis oligolepis snakes can be used as safe and natural sources for the development of new AMPs potent against microorganisms of clinical and scientific interest.

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.

Les sources hydrothermales océaniques profondes renferment des écosystèmes extrêmes, situés dans la zone abyssale des Océans. Dans ces environnements dépourvus de lumière, la production primaire est réalisée par la chimiosynthèse microbienne. Ces milieux sont colonisés par des espèces animales, dont la plupart vivent en associations plus ou moins fortes avec des micro-organismes. La crevette Rimicaris exoculata est une espèce hydrothermale endémique des sites de la Ride-Médio-Atlantique (MAR), qui domine la plupart des sites qu’elle colonise. Ce crustacé a pour particularité de posséder deux communautés symbiotiques : une située dans son céphalothorax hypertrophié et une inféodée à son tractus digestif. Tout d’abord, ce travail de thèse s’est concentré sur l’étude de la communication bactérienne (Quorum Sensing ou QS) au sein des communautés ectosymbiotiques de R. exoculata au cours de son cycle de mue et de vie. Ensuite, ce travail s’est focalisé sur l’identification d’un peptide antimicrobien (PAM), puis à rechercher sa fonction dans l'immunité et le contrôle des symbiotes chez Rimicaris exoculata. Ce travail a permis, d’une part, de confirmer la présence de deux gènes du QS (luxS et luxR) dans les communautés ectosymbiotiques de R. exoculata sur quatre sites hydrothermaux : Rainbow, TAG, Snake Pit et Logatchev. Ces gènes étant plus divergents que ceux de l'ARNr 16S, leur utilisation comme marqueurs génétiques biogéographiques pour retracer l'origine des individus est discuté. Ce travail a permis, d’autre part, d’identifier pour la première fois un PAM (sus nommé Re-crustin), chez un arthropode hydrothermal. Les données suggèrent une participation de ce PAM dans le contrôle de l’ectosymbiose. L’ensemble de ces travaux apporte de nouvelles hypothèses sur l’interaction entre les épibiontes du céphalothorax et la crevette Rimicaris exoculata. / Deprived of light, the deep-sea hydrothermal vents are extremes ecosystems sustained by micobial chemosynthesis. These environments are colonized by animal species living in close relationships with these chemoautotrophic micro-organisms, eating them or establishing long term interactions with them, may they be trophic or not only. The shrimp Rimicaris exoculata is an endemic hydrothermal species of the Mid-Atlantic Ridge (MAR) sites. This crustacean represents the predominant macrofauna of some sites of the MAR. It lives in symbiotic association with two distinct microbial communities qualified as ectosymbiosis. One is located in its gill chamber and one in its gut. First, this work focused on the study of bacterial communication (Quorum Sensing or QS) within the ectosymbiontic communities during the molting and life cycles of R. exoculata. Then, we focused on an antimicrobial peptide (AMP) identification and search for its function in R. exoculata immunity and in controlling symbionts. Two QS genes (luxS and luxR) were identified in the R. exoculata ectosymbiontic community at different shrimp molt stages and life stages at the Rainbow, TAG, Snake Pit and Logatchev vent sites.As these genes are more divergent than that of 16S rRNA, they could be then used as biogeographical genetic markers tools to trace back the origin of individuals to a location or between locations along its life cycle. This work reports also the first description of an AMP in an extremophile arthropod (namely Recrustin). Data suggest a participation of this AMP in the control of the ectosymbiosis in Rimicarisexoculata. All this work provides new hypotheses wich are discussed in the manuscript, dealing with the interaction between symbionts and Rimicaris exoculata.

The world-wide transdermal drug delivery market is quite large, but only a small number of agents have FDA approval. The primary reason for such limited development is the difficulty in permeating the stratum corneum layer of human skin. In our study, we developed a novel percutaneous delivery enhancing approach. Magainin peptide was previously shown to disrupt vesicles from stratum corneum lipid components and this ability of magainin allows us to propose that magainin can increase skin permeability. Therefore, we tested the hypothesis that magainin, a pore-forming peptide, can increase skin permeability by disrupting stratum corneum lipid structure and that magainin¡¯s enhancement requires co-administration of a surfactant chemical enhancer to increase magainin penetration into the skin. In support of these hypotheses, synergistic enhancement of transdermal permeation can be observed with magainin peptide in combination of N-lauroyl sarcosine (NLS) in 50% ethanol-PBS solution. The exposure to NLS in 50% ethanol solution increased in vitro skin permeability to fluorescein 15 fold and the addition of magainin synergistically increased skin permeability 47 fold. In contrast, skin permeability was unaffected by exposure to magainin without co-enhancement by NLS-ethanol. To elucidate the mechanism of this synergistic effect, several characterization methods such as differential scanning calorimetry, Fourier transform infrared spectroscopy, and X-ray diffraction were applied. These analyses showed that NLS-ethanol disrupted stratum corneum lipid structure and that the combination of magainin and NLS-ethanol disrupted stratum corneum lipids even further. Furthermore, confocal microscopy showed that magainin in the presence of NLS-ethanol penetrated deeply and extensively into stratum corneum, whereas magainin alone penetrated poorly into the skin. Together, these data suggest that NLS-ethanol increased magainin penetration into stratum corneum, which further increased stratum corneum lipid disruption and skin permeability. Finally, skin permeability was enhanced by changing the charge of magainin peptide via pH change. We modulated pH from 5 to 11 to change the magainin charge from positive to neutral, which decreased skin permeability to a negatively charged fluorescein and increased skin permeability to a positively charged granisetron. This suggests that an attractive interaction between the drug and magainin peptide improves transdermal flux.

Bacterial pathogens assemble complex surface structures in an attempt to circumvent host immune detection. A great example is the glycolipid known as lipopolysaccharide or lipooligosaccharide (LPS), the major surface molecule in nearly all gram-negative organisms. LPS is anchored to the bacterial cell surface by a anionic hydrophobic lipid known as lipid A, the major agonist of the mammalian TLR4-MD2 receptor and likely target for cationic antimicrobial peptides (CAMPs) secreted by host cells (i.e. defensins). In this work we investigate LPS modification machinery in related ε-proteobacteria, Helicobacter pylori and Campylobacter jejuni, two important human pathogens, and demonstrate that enzymes involved in LPS modification not only play a role in evasion of host defenses but also an unexpected role in bacterial locomotion. More specifically, we identify the enzyme responsible for 4'-dephosphorylation of H. pylori lipid A, LpxF. Demonstrating that lipid A depohsphorylation at the 1 and 4'-positions by LpxE and LpxF, respectively, are the primary mechanisms used by H. pylori for CAMP resistance, contribute to attenuated TRL4-MD2 activation and are required for colonization of a the gastric mucosa in murine host. Similarly in C. jejuni, we identify an enzyme, EptC, responsible for modification of lipid A at both the 1 and 4'-positions with phosphoethanolamine (pEtN), also required for CAMP resistance in this organism. Suprisingly, EptC was found to serve a dual role in modifying not only lipid A with pEtN but also the flagellar rod protein FlgG at residue Thr75, required for motility and efficient flagella production. This work links membrane biogenesis with flagella assembly, both shown to be required for colonization of a host and adds to a growing list of post-translational modifications found in prokaryotes. Understanding how pathogens evade immune detection, interphase with the surrounding environment and assemble major surface features is key in the development of novel treatments and vaccines. / text

Alzheimer’s Disease (AD) is a chronic progressive neurological condition, clinically characterized by memory deficits, cognitive and physical impairment, and personality changes. Traditionally, AD was considered a type of protein folding disorder. Here, the concept of AD as an autoimmune disease of the innate immune system was developed. After exploring evolutionary connections between the AD peptide β-amyloid (Aβ) and known antimicrobial peptides (AMPs), and elucidating the structural similarities between Aβ and AMPs, a mechanism of action for Aβ’s antimicrobial activity is proposed that is based on the compromise of bacterial membranes. Following these theoretical considerations, experimental evidence is presented for the production of Aβ by cells in response to infection, and for Aβ’s antibacterial and antiviral activity. Rooted in similarities of the cell membranes of neuronal and bacterial cells in terms of lipid composition and transmembrane potential, it is hypothesised that Aβ’s neurotoxicity is caused by its misguided attack on neurons as an AMP. In reversing the concept of Aβ as an AMP, the similarity of AMPs to Aβ is demonstrated in experiments revealing the neurotoxicity of two AMPs, LL 37, and cecropin A. To determine a mechanism for the progressive nature of AD, it was shown that, although apoptosis may be involved in AD, it is actually necrosis that is responsible for the propagation of neuronal cell death so characteristic of AD. With the Vicious Cycle of AD, a scheme was devised, integrating the results obtained here with data and research from other groups, which explains the chronic and progressive nature of AD as a result of Aβ’s physiological role as an AMP and innate immune system effector. Borne from Aβ’s activity as an AMP and its central role in the Vicious Cycle of AD, a question was investigated: do antibiotics, such as penicillin, that cause release of bacterial endotoxins due to their mechanism of action, trigger the Vicious Cycle of AD and thus lead to the development of AD? Preliminary evidence supporting this notion was presented.

Les objectifs de cette thèse étaient d'explorer les mécanismes moléculaires et les bases génétiques impliquées dans la survie des huîtres Crassostrea gigas à des maladies infectieuses, en considérant deux souches de Vibrio pathogènes pour l'huître (V. splendidus LGP32 et V. aestuarianus LPi 02/41) qui ont été associées aux phénomènes de mortalités massives d'huîtres en France. Par l'approche transcriptomique de « Digital Gene Expression », nous avons identifié des composants génétiques d'une réponse efficace à des infections par des Vibrio virulents. La capacité de survie des huîtres se traduit par l'expression basale d'une combinaison de 14 gènes hémocytaires, une signature de survie, et par l'induction de différentes fonctions cellulaires au cours de la réponse immunitaire. Une analyse transcriptomique détaillée au niveau individuel a révélé un extraordinaire polymorphisme d'expression basale des gènes, incluant des cas où chez certaines huîtres des transcrits sont absents. Afin de comprendre ce polymorphisme, nous nous sommes intéressés à la caractérisation d'une nouvelle famille de peptides antimicrobiens (PAMs), les big défensines (Cg-BigDef). Nous avons montré que Cg-BigDef est une famille de PAMs composée de trois membres et diversifiée en termes de séquences, d'organisation génomique et de régulation de l'expression des gènes. Les Cg-BigDefs sont codées par des gènes distincts dont l'expression est régulée suivant différents modes en réponse à une infection. Chose intéressante, certaines huîtres n'expriment pas simultanément les trois formes de Cg-BigDef ou dans certains cas, n'en expriment aucune. Nous avons démontré que l'absence d'expression basale de Cg-BigDef est liée à l'absence de gène correspondant dans le génome des huîtres. C'est la première mise en évidence chez un invertébré de variation de présence/absence (PAV) de gènes, un phénomène qui pourrait contribuer à une susceptibilité accrue aux maladies infectieuses. / The objectives of this thesis were to explore the molecular mechanisms and genetic bases involved in Crassostrea gigas oyster survival to infectious diseases, considering two Vibrio strains (V. splendidus LGP32 and V. aestuarianus LPi 02/41) pathogenic for oysters which have been shown to be involved in C. gigas mass mortalities in France. By the Digital Gene Expression transcriptomic approach, we have identified some genetic components implicated in a successful response and survival to virulent Vibrio infections. Oyster survival capacity is reflected by the basal expression of a selected combination of hemocyte genes, a 14-gene survival signature, and by the induction of some cellular functions during the oyster immune response. A detailed transcriptomic analysis at individual level revealed an extraordinary interindividual polymorphism in basal gene expression, including cases where some transcripts are fully absent. In order to understand this striking variability in gene expression, we have focused on the characterization of a novel family of antimicrobial peptides (AMP) in C. gigas oysters, the big defensins (Cg-BigDef). We have shown that Cg-BigDef is an AMP family, composed of three members, and diversified in terms of sequences but also in terms of genomic organization and regulation of gene expression. Each Cg-BigDef form is encoded by a distinct gene that follows different patterns of gene regulation upon Vibrio infection. Interestingly, some oysters were shown do not express simultaneously the three Cg-BigDef forms or any Cg-BigDef. We demonstrated that the absence of Cg-BigDef basal gene expression is likely due to the absence of the Cg-bigdef gene in oyster genome. This is the first evidence in an invertebrate of a presence/absence variation (PAV) of genes, a phenomenon that could be associated to a susceptibility to infectious diseases.

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.