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Antimicrobial peptides (AMPs) have potential antifungal activities, however, their intracellular protein targets are poorly reported. Proteome microarray is an effective tool with high-throughput and rapid platform that systematically identifies the protein targets. In this study, we have used yeast proteome microarrays for systematical identification of the yeast protein targets of Lactoferricin B (Lfcin B) and Histatin-5. A total of 140 and 137 protein targets were identified from the triplicate yeast proteome microarray assays for Lfcin B and Histatin-5, respectively. The Gene Ontology (GO) enrichment analysis showed that Lfcin B targeted more enrichment categories than Histatin-5 did in all GO biological processes, molecular functions, and cellular components. This might be one of the reasons that Lfcin B has a lower minimum inhibitory concentration (MIC) than Histatin-5. Moreover, pairwise essential proteins that have lethal effects on yeast were analyzed through synthetic lethality. A total of 11 synthetic lethal pairs were identified within the protein targets of Lfcin B. However, only three synthetic lethal pairs were identified within the protein targets of Histatin-5. The higher number of synthetic lethal pairs identified within the protein targets of Lfcin B might also be the reason for Lfcin B to have lower MIC than Histatin-5. Furthermore, two synthetic lethal pairs were identified between the unique protein targets of Lfcin B and Histatin-5. Both the identified synthetic lethal pairs proteins are part of the Spt-Ada-Gcn5 acetyltransferase (SAGA) protein complex that regulates gene expression via histone modification. Identification of synthetic lethal pairs between Lfcin B and Histatin-5 and their involvement in the same protein complex indicated synergistic combination between Lfcin B and Histatin-5. This hypothesis was experimentally confirmed by growth inhibition assay.

Made available in DSpace on 2018-12-11T17:02:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-09-01 Objective Controversies exist regarding the relationship between the concentrations of antimicrobial peptides (AMPs) and presence of dental caries in children. Thus, the aim of this study was to examine levels of AMPs in saliva of caries-free (CF), early childhood caries (ECC) and severe early childhood caries (S-ECC) children to determine if the levels of these salivary peptides individually or in combinations were related to caries severity and mutans streptococci levels. Design 36 to 60 month-old children were selected to participate in this study. Children were grouped into CF group (n = 29), ECC group (n = 25) and S-ECC group (n = 29). Saliva was collected from children for microbiological analysis by culture. Salivary concentrations of cathelicidin LL-37, human β-defensin 2 (hBD-2), human β-defensin 3 (hBD-3) and histatin-5 (HTN-5) were determined by ELISA. Results Salivary concentrations of AMPs did not differ among CF, ECC and S-ECC groups. Data showed positive correlations between mutans streptococci levels and salivary hBD-2 or HTN-5. Positive correlations were found between hBD-2, hBD-3, LL-37 and HTN-5. Combinations among AMPs, mainly LL-37, were positively associated with caries levels. Conclusions Salivary concentrations of AMPs individually were not associated with the severity of early childhood caries. The stimulus of caries appears to trigger a biological response, however, with a combination of these peptides. UNESP-Univ. Estadual Paulista Department of Pediatric Dentistry and Public Health Araçatuba Dental School, Rua José Bonifácio, 1193 Forsyth Institute Department of Microbiology Harvard University Harvard School of Dental Medicine UNESP-Univ. Estadual Paulista Department of Pediatric Dentistry and Public Health Araçatuba Dental School, Rua José Bonifácio, 1193

Candida albicans is a common microorganism of human’s microbiota and can be easily found in both respiratory and gastrointestinal tracts as well as in the genitourinary tract. Approximately 30% of people will be infected by C. albicans during their lifetime. Due to its easy adaptation, this microorganism started to present high resistance to antifungal agents which is associated with their indiscriminate use. There are several reports of adaptive mechanisms that this species can present. Some of them are intrinsic alteration in drug targets, secretion of extracellular enzymes to promote host protein degradation and efflux receptors that lead to a diminished action of common antifungal and host’s innate immune response. The current review aims to bring promising alternatives for the treatment of candidiasis caused mainly by C. albicans. One of these alternatives is the use of antifungal peptides (AFPs) from the Histatin family, like histatin‐5. Besides that, our focus is to show how nanotechnology can allow the application of these peptides for treatment of this microorganism. In addition, our intention is to show the importance of nanoparticles (NPs) for this purpose, which may be essential in the near future.

Submitted by Carolina Reis Zambom null (carolinarz@gmail.com) on 2018-03-20T17:16:52Z No. of bitstreams: 1 Dissertação Carol_versão correçãotodos_versãofinalpósdefesa-edit.pdf: 2874016 bytes, checksum: 09ff8b1558d91035a2e106afc33f8bd8 (MD5) Approved for entry into archive by Ana Carolina Gonçalves Bet null (abet@iq.unesp.br) on 2018-03-23T17:38:22Z (GMT) No. of bitstreams: 1 zambom_cr_me_araiq_int.pdf: 2737311 bytes, checksum: 4eca302db8e9f1efe9d15f6049d7aa9a (MD5) Made available in DSpace on 2018-03-23T17:38:22Z (GMT). No. of bitstreams: 1 zambom_cr_me_araiq_int.pdf: 2737311 bytes, checksum: 4eca302db8e9f1efe9d15f6049d7aa9a (MD5) Previous issue date: 2018-03-02 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Atualmente aproximadamente 75-88% das infecções fúngicas são causadas por espécies de Candida, que geram um custo de 1,7 bilhões de dólares para a saúde pública, somente nos EUA. Candida albicans é o principal microrganismo causador da candidíase bucal, uma infecção da mucosa oral do organismo humano. A patogenicidade dessa espécie está relacionada com a formação de biofilmes, que agem como um revestimento impermeável e protetor, e tornam o microrganismo resistente a medicamentos convencionais. Neste caso, os antifúngicos mais comumente utilizados, não apresentam ação eficaz. Por esse motivo, a busca por novas opções de tratamento e por novos medicamentos está em constante desenvolvimento, principalmente na área da biotecnologia. Um exemplo disso são os peptídeos antifúngicos da família das Histatinas, entre eles a Histatina-5. Trata-se de um peptídeo naturalmente encontrado na saliva humana, mas que sofre rápida degradação quando presente na cavidade bucal, que é seu local de ação. Diante disto, o objetivo deste trabalho é o desenvolvimento de lipossomas de diferentes composições lipídicas, na intenção de proteger o peptídeo e melhorar sua ação, preservando seu potencial antifúngico. Para isso foi sintetizado o peptídeo 0WHistatina-5, um análogo do peptídeo Histatina-5, que contém o aminoácido triptofano em sua sequência. Foi utilizado o método de síntese em fase sólida, seguido de clivagem e purificação, com confirmação da massa molecular utilizando HPLC e ESI-MS. Os lipossomas foram produzidos pelo método de hidratação do filme lipídico, em 3 composições lipídicas diferentes, denominadas de F1, F2 e F3. F1 possui somente DPPC e Chol em sua composição, F2 contém, além desses componentes, PEG e F3 contém DPPC, Chol e POPG. Os lipossomas foram submetidos a processo de extrusão e sonicação para padronização do tamanho das vesículas, e estudo da melhor técnica para sua produção. Os lipossomas foram caracterizados por espalhamento de luz dinâmico determinação da eficiência de encapsulação, cinética de liberação, estabilidade e avaliação da atividade antifúngica. O método de síntese do peptídeo 0WHistatina-5 foi adequado e o processo de purificação possibilitou a obtenção do peptídeo com alto grau de pureza. Os lipossomas obtidos por extrusão apresentaram tamanho médio na faixa de 100 nm, enquanto os lipossomas obtidos por sonicação apresentaram tamanho menor, na faixa de 90 nm. Os lipossomas contendo 0WHistatina-5, apresentaram aumento em seu tamanho médio, indicando que o peptídeo está contido no compartimento interno aquoso dos lipossomas. A eficiência de encapsulação foi maior para os lipossomas obtidos por sonicação, sendo de 34,5% para a formulação F1, que contém DPPC e Chol em sua composição. A composição lipídica dos lipossomas está relacionada com a sua eficiência de encapsulação, e a presença de colesterol dificultou a encapsulação do peptídeo. A estabilidade das formulações também está relacionada com sua composição, sendo a formulação F3, obtida por sonicação e com POPG em sua formulação, a que apresentou melhor estabilidade quando armazenada por 60 dias a temperatura de 4°C. As formulações desenvolvidas apresentaram capacidade de liberar o peptídeo pelo tempo total de 96 horas, com o primeiro pico de liberação após 5 horas, e novo aumento do conteúdo liberado após 30 horas. O ensaio antimicrobiano foi realizado utilizando C. albicans ATCC 10231, e demostrou que no tempo de 4 horas a inibição para F1 foi de 66,5%. Assim, este trabalho demonstrou que a utilização de sistemas nanoestruturados é de grande importância para viabilizar a aplicação do peptídeo Histatina-5 em estudos terapêuticos, e em estudos in vivo no futuro. Currently, approximately 75-88% of fungal infections are caused by Candida species, which generate a cost of $ 1.7 billion for public health in the US. Candida albicans is the main microorganism that causes oral candidiasis, an infection of the oral mucosa of the human organism. The pathogenicity of this species is related to the formation of biofilms, which act as an impermeable and protective coating, and make the microorganism resistant to conventional drugs. In this case, the most commonly used antifungals, have no effective action. For this reason, the search for new treatment options and new drugs is in constant development, especially in the biotechnology area. The antifungal peptides of the Histatin family, including Histatin-5, are an example. The Histatin-5 is a peptide naturally found in human saliva, but it undergoes rapid degradation when present in the oral cavity, which is its place of action. For this reason, this work aimed at developing liposomes of different lipid compositions, in order to protect the peptide, improve its action, and preserve its antifungal potential. For this, the peptide 0WHistatin-5, an analog of the peptide Histatin-5, was synthesized, which contains the amino acid tryptophan in its sequence. Therefore, the peptide was synthesized manually by the solid phase synthesis method, followed by cleavage and purification. The molecular weight was confirmed by using HPLC and ESI-MS. The liposomes were produced by the lipid film hydration method, with three different lipid compositions, named F1, F2 and F3. F1 has only DPPC and Chol in its composition, F2 contains, in addition to these components, PEG and F3 contains DPPC, Chol and POPG. The liposomes were submitted to extrusion and sonication processes, in order to standardize their vesicle size, and determine the best technique for their production. The dynamic light scattering technique was used to characterize the liposomes, which were tested for encapsulation efficiency, release kinetics, stability and evaluation of the antifungal activity. The synthesis method of the Histatin-5 was adequate and the purification process allowed to obtain the peptide in high purity. The liposomes obtained by extrusion presented average size in the range of 100 nm, while the liposomes obtained by sonication presented a smaller size, in the range of 90 nm. Liposomes loaded with Histatin-5 showed an increase in their mean size, which indicated that the peptide was contained in the intern aqueous compartment of the liposomes. The encapsulation efficiency was higher for the liposomes obtained by sonication. The F1 formulation presented an encapsulation efficiency of 34.5%, which contains DPPC and Chol in its composition. The lipid composition of the liposomes was related to their encapsulation efficiency, and the presence of cholesterol hindered the encapsulation of the peptide. The stability of the formulations was also related to their compositions. The F3 formulation obtained by sonication and containing POPG presented a higher stability when stored for 60 days at 4°C. The formulations showed the ability to release the peptide in the total period of 96 hours. The first release peak was observed after 5 hours, and a further increase of the released content was verified after 30 hours. The antimicrobial assay was performed using C. albicans ATCC 10231. It demonstrated that the inhibition for F1 was 66.5% after four hours of incubation, and it was maintained at 30% until the end of the experiment. Thus, this work conclusions showed that the use of nanostructured systems is of great importance to enable the application of Histatin-5 in therapeutic studies, and in vivo studies in the future. 132393/20166

Background: Recently, a continuous growth of interest has been observed in antimicrobial peptides (AMPs) in the light of an alarming increase in resistance of bacteria and fungi against antibiotics. AMPs are used as biomarkers in diagnosis and monitoring of oral cavity pathologies. Therefore, the determination of specific protein profiles in children diagnosed with early childhood caries (ECC) might be a basis for effective screening tests and specialized examinations which may enable progression of disease. Methods: The objective of the studies was to determine the role of histatin-5 and $\beta$-defensing-2 as a diagnostic marker of early childhood caries progression. In this work, results of concentration determination of two salivary proteins (histatin-5 and $\beta$-defensin-2) were presented. In addition, bacterial profiles from dental plaque in various stages of ECC and control were marked. The assessment of alteration in the concentration of these two proteins in a study group of children with various stages of ECC and a control group consisting of children with no symptoms was performed by enzyme-linked immunosorbent assays. Results: The statistical analysis showed a significant increase in the concentration of histatin-5 and $\beta$-defensin-2 in the study group compared to the control group and correlated with the progression of the disease. Conclusions: The confirmation of concentration changes in these proteins during the progression of dental caries may discover valuable disease progression biomarkers.

P(論文), Histatin-5 and the basic Proline-rich peptide P-E (PRP-PE) have been revealed as candidates of bitter binding proteins. However, it has not been confirmed whether these compounds were carriers of or blockers to bitter taste so far. On the other hand, it has been reported that psychical stress reduced the sensitivity of bitterness. The present study was carried out to measure the clinical concentrations of Histatin-5 and PRP-PE in saliva under the mental stress. Stimulated parotid saliva was obtained from the parotid glands of 10 healthy adult subjects before and after the load of stress of the Kraepelin psycho-diagnostic test with a loud noise (5,000Hz, 100dB) for 30 mins. The salivary cortisol concentration and the LF/HF ratio on ECG were adopted as indices of sympathetic nervous activity to check a liability for the load of stress. The concentrations of Histatin-5 and PRP-PE in saliva were quantified by ELISA test. A significant increase in the salivary cortisol after the stress was recognized in half of the subjects, among which an increase of LF/HF ratio was also observed. Histatin-5 in the parotid saliva was significantly reduced by the stress in this group, but PRP-PE was not. These findings suggest that Histatin-5 in saliva might be a carrier protein of bitter substances, but a function of PRP-PE was not clear in this study.

In order to prevent biofilm formation by Candida albicans, several cationic peptides were covalently bound to polydimethylsiloxane (PDMS). The salivary peptide histatin 5 and two synthetic variants (Dhvar 4 and Dhvar 5) were used to prepare peptide functionalized PDMS using 4-azido-2,3,5,6-tetrafluoro-benzoic acid (AFB) as an interlinkage molecule. In addition, polylysine-, polyarginine-, and polyhistidine-PDMS surfaces were prepared. Dhvar 4 functionalized PDMS yielded the highest reduction of the number of C. albicans biofilm cells in the Modified Robbins Device. Amino acid analysis demonstrated that the amount of peptide immobilized on the modified disks was in the nanomole range. Poly-d-lysine PDMS, in particular the homopeptides with low molecular weight (2500 and 9600) showed the highest activity against C. albicans biofilms, with reductions of 93% and 91%, respectively. The results indicate that the reductions are peptide dependent.

Histatin-5 is a peptide secreted in the human saliva, which possesses powerful antifungal activity. Previous studies have shown that this peptide exerts its candidacidal activity, through the inhibition of both mitochondrial respiration and the formation of reactive oxygen species. The purpose of the present study was to investigate the biological consequences of histatin-5 action on mammalian mitochondria to verify if the toxic mechanism exerted on mitochondria from Candida albicans is an exclusive for fungal cells. Moreover, hypothesising that the damage exerted on mitochondria may induce programmed cellular death pathways, we evaluated two main markers of apoptosis: the mitochondrial membrane potential (DeltaPsi) and the release of cytochrome c. The results obtained show that exposure of isolated mammalian mitochondria to histatin-5 determines: (i) a large inhibition of the respiratory chain at the level of complex I, (ii) a slight decrease in the mitochondrial membrane potential, and (iii) no release of cytochrome c.

Background Histatins are histidine rich polypeptides produced in the parotid and submandibular gland and secreted into the saliva. Histatin-3 and −5 are the most important polycationic histatins. They possess antimicrobial activity against fungi such as Candida albicans. Histatin-5 has a higher antifungal activity than histatin-3 while histatin-3 is mostly involved in wound healing in the oral cavity. We found that these histatins, like other polycationic peptides and proteins, such as LL-37, lysozyme and histones, interact with extracellular actin. Results Histatin-3 and −5 polymerize globular actin (G-actin) to filamentous actin (F-actin) and bundle F-actin filaments. Both actin polymerization and bundling by histatins is pH sensitive due to the high histidine content of histatins. In spite of the equal number of net positive charges and histidine residues in histatin-3 and −5, less histatin-3 is needed than histatin-5 for polymerization and bundling of actin. The efficiency of actin polymerization and bundling by histatins greatly increases with decreasing pH. Histatin-3 and −5 induced actin bundles are dissociated by 100 and 50 mM NaCl, respectively. The relatively low NaCl concentration required to dissociate histatin-induced bundles implies that the actin-histatin filaments bind to each other mainly by electrostatic forces. The binding of histatin-3 to F-actin is stronger than that of histatin-5 showing that hydrophobic forces have also some role in histatin-3- actin interaction. Histatins affect the fluorescence of probes attached to the D-loop of G-actin indicating histatin induced changes in actin structure. Transglutaminase cross-links histatins to actin. Competition and limited proteolysis experiments indicate that the main histatin cross-linking site on actin is glutamine-49 on the D-loop of actin. Conclusions Both histatin-3 and −5 interacts with actin, however, histatin 3 binds stronger to actin and affects actin structure at lower concentration than histatin-5 due to the extra 8 amino acid sequence at the C-terminus of histatin-3. Extracellular actin might regulate histatin activity in the oral cavity, which should be the subject of further investigation. Electronic supplementary material The online version of this article (doi:10.1186/s12858-017-0078-0) contains supplementary material, which is available to authorized users.

The mechanisms of adhesion to solid surfaces enable S. mutans to colonize oral cavities and form biofilms, which play an important role in caries development. Additional properties enabling the survival of S. mutans in the oral cavity include its ability to survive in acidic environments and specific interactions with other microorganisms inhabiting this ecosystem.The aim of this study was to determine the antibacterial activity of saliva histatin-5 (peptide) and lysozyme (protein) against S. mutans and L. rhamnosus, as representatives of physiological flora.The study involved strains of physiological (L. rhamnosus) and cariogenic (S. mutans) flora isolated from one patient with diagnosed early caries of the deciduous teeth.It was proved that the presence of probiotic L. rhamnosus bacteria in the environment had a negative impact on the ability of S. mutans to produce biofilm. Moreover, the antibacterial activity of histatin-5 was confirmed, and it inhibited S. mutans growth at concentrations of 27.2 μg/ml and 54.4 μg/ml, both individually and in a mixture with lysozyme (in a total concentration of 54.4 μg/ml).The data obtained constitute a promising result due to their potential future application in the prevention and early diagnosis of caries.