Genetic analysis and virulence factors of clinical isolates of Candida albicans from patients with chronic periodontitis with diabetes mellitus type II

Orientadores: Reginaldo Bruno Gonçalves, Cristiane DuqueTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Candida spp. são leveduras comensais que habitam diferentes sítios da cavidade bucal. Em indivíduos saudáveis, sem alterações imunológicas, esses microrganismos não causam doença. Entretanto, diante de condições imunossupressoras, essas leveduras podem se tornar mais virulentas e expressar patogenicidade. Espécies de Candida apresentam diversos fatores de virulência, incluindo mecanismos de adesão e invasão celular associado à produção de enzimas que auxiliam na degradação tecidual e facilitam sua proliferação na mucosa bucal. Estudos têm demonstrado a presença de Candida sp. em sítios periodontais de pacientes com periodontite crônica, principalmente quando estes são imunologicamente comprometidos. Entretanto, ainda é desconhecido o papel desses microrganismos na patogênese da doença periodontal. Os objetivos do presente trabalho foram: 1) identificar a presença de espécies de Candida e periodontopatógenos por PCR em sítios bucais de pacientes diabéticos ou não com periodontite crônica; 2) isolar cepas de Candida albicans desses pacientes e avaliá-las quanto à atividade das enzimas proteinase, fosfolipase e hemolisina e os graus de hidrofobicidade da superfície celular, sob diferentes condições atmosféricas, além de realizar a análise genotípica desses isolados; 3) avaliar a capacidade de adesão e invasão de cepas de Candida albicans com diferentes graus de hidrofobicidade, em fibroblastos gengivais humanos. As reações de PCR mostraram que os diabéticos tiveram maior prevalência de Candida spp. principalmente C. albicans e C. dubliniensis, e menor freqüência de Tannerella forsythia, quando comparado aos pacientes não diabéticos, para bolsa periodontal e furcas. C. glabrata e C. tropicalis não foram encontradas em sítios periodontais de pacientes não diabéticos. Dos pacientes diabéticos, foram isoladas 128 cepas de C. albicans, das quais 51.6% foram determinadas como genótipo B e 48.4% como genótipo A. As condições ambientais consideradas neste estudo, níveis reduzidos de oxigênio ou anaerobiose, não modificaram o tipo de hemólise realizado pelo microrganismo, sendo que a maioria das cepas foi alfa-hemolítica. Nesses ensaios, 100% das cepas em anaerobiose apresentaram as colônias rugosas, enquanto que em ambiente de oxigênio reduzido, houve variação em relação à morfologia e a maioria delas apresentou colônia lisa. Com relação à atividade de proteinase e fosfolipase, cepas de C. albicans não produziram as enzimas na ausência total de oxigênio. Em ambiente com nível reduzido de oxigênio, a maioria das cepas de C. albicans foram fortemente produtoras de proteinase e a maioria das cepas foi positiva para fosfolipase. A hidrofobicidade foi mais alta na condição de anaerobiose. A partir desses resultados, foram selecionadas 16 cepas com alta ou baixa hidrofobicidade e avaliadas quanto à capacidade de adesão e invasão em fibroblastos gengivais humanos. Foi verificado que ambos os processos foram maiores nas cepas com alta hidrofobicidade. A produção de óxido nítrico foi maior para as cepas mais hidrofóbicas. Os resultados demonstraram que as espécies de Candida podem ser encontradas, em grande proporção, em bolsas periodontais e furcas de pacientes portadores de periodontite crônica, principalmente naqueles acometidos por diabetes mellitus. A maioria das cepas de C. albicans apresentou atividade enzimática, que atuaria diretamente na degradação tecidual. Além disso, a hidrofobicidade das cepas de C. albicans mostrou estar relacionada à maior capacidade de adesão e invasão em fibroblastos. Todos esses fatores de virulência aumentam a patogenicidade da Candida, que poderia colaborar na progressão da doença periodontal, principalmente em pacientes imunodeficientesAbstract: Candida spp. are commensal yeasts that inhabit different sites of the oral cavity. In healthy subjects, without immunological alterations, these microorganisms do not cause disease. However, in immunosuppressive conditions, these yeasts can become more virulent and express pathogenicity. Candida species have different virulence factors, including mechanisms of cell adhesion and invasion associated with the production of enzymes that facilitate tissue degradation and their proliferation in oral mucosa. Studies have shown the presence of Candida spp. in periodontal sites of patients with chronic periodontitis, especially when they are immunologically compromised. However is still unknown their role in the pathogenesis of periodontal disease. The objectives of this study were: 1) to identify the presence of Candida species and putative periodontopathogens by PCR in periodontal sites of diabetic or non-diabetic patients with chronic periodontitis; 2) to isolate strains of Candida albicans in these patients and evaluate the proteinase, phospholipase and haemolysin activities and degrees of cell surface hydrophobicity under different atmospheric conditions, besides to performe the genotypic analysis of these isolates; 3) to evaluate the ability of adhesion and invasion of Candida albicans strains with different degrees of hydrophobicity, in human gingival fibroblasts. The PCR reactions revealed that diabetics had higher prevalence of Candida spp., mainly C. albicans and C. dubliniensis, and lower T. forsythia frequency, when compared to non-diabetic patients, for both periodontal sites. C. glabrata and C. tropicalis were not found in periodontal pockets and furcation sites of non-diabetic patients. From diabetic patients, it was isolated 128 strains of C. albicans and 51.6% were determined as genotype B and 48.4% as genotype A. The atmospheric conditions, reduced oxygen and anaerobiosis, did not change the type of hemolysis, and the most of strains were alpha-hemolytic. From these assays, 100% of the strains under anaerobiosis showed rough colonies, whereas in an environment with reduced oxyen was no change in relation to morphology and most of them had smooth colony. Considering proteinase and phospholipase activities, C. albicans strains did not produce the enzymes in the total absence of oxygen. In reduced oxygen, the majority of C. albicans strains were strong proteinase producers and most strains were positive for phospholipase. Hydrophobicity was higher in anaerobic condition. From these results, 16 hydrophobic or hydrophilic strains were selected and evaluated their ability of adhesion and invasion in human gingival fibroblasts. Both processes were greater in strains with high hydrophobicity. The production of nitric oxide was higher for hydrophobic strains. The results showed that Candida species can be found in large proportion, in periodontal pockets and furcation of patients with chronic periodontitis, especially diabetics. The most of C. albicans strains showed enzymatic activity, which could act directly on tissue degradation. Moreover, the hydrophobicity of C. albicans seems to be related to higher capacity of adhesion and invasion in fibroblasts. All these virulence factors enhance the pathogenicity of Candida that could collaborate for the progression of periodontal diseaseDoutoradoMicrobiologia e ImunologiaDoutor em Biologia Buco-Denta

Alternative animal and non-animal models for drug discovery and development: bonus or burden?

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOMammalian models have served as a basis for R&D over the past decades. Nevertheless, these models are expensive, laborious, may yield results that cannot always be translated into the human in vivo situation and, more recently, have reverberated great social and ethical dilemmas. Hence, the prospect of changes in the global scientific scenario and the Three Rs principle (Reduction, Replacement and Refinement) have encouraged the development of alternative methods to the use of mammals. Despite the efforts, suitable alternative tests are not available in all areas of biomedical research, as regulatory acceptance requires time, prior validation and robust financial and scientific investment. In this perspective, we aim to shed light on the concepts, challenges and perspectives for implementation of innovative alternative animal and non-animal methods in scientific research. The applicability and meaningfulness of invertebrate animal models, in silico analysis and reverse pharmacology are discussed, among other aspects of relevance in today's scenario. Overall, the use of alternative models, including Artemia salina (brine shrimp), Caenorhabditis elegans (roundworm), Danio rerio (zebra fish), Drosophila melanogaster (fruit fly), Galleria mellonella (greater waxmoth) and in silico modelling, increased 909% from 1990 to 2015, as compared to 154% of conventional mammals in the same period. Thus, technological and scientific advancements in the fields of toxicology and drug development seem to have diminished the need for mammalian models. Today, however, mammals still remain critically indispensable to provide - in most cases -reliable data subsidizing and validating translation into the clinical setting.Mammalian models have served as a basis for R&D over the past decades. Nevertheless, these models are expensive, laborious, may yield results that cannot always be translated into the human in vivo situation and, more recently, have reverberated great social and ethical dilemmas. Hence, the prospect of changes in the global scientific scenario and the Three Rs principle (Reduction, Replacement and Refinement) have encouraged the development of alternative methods to the use of mammals. Despite the efforts, suitable alternative tests are not available in all areas of biomedical research, as regulatory acceptance requires time, prior validation and robust financial and scientific investment. In this perspective, we aim to shed light on the concepts, challenges and perspectives for implementation of innovative alternative animal and non-animal methods in scientific research. The applicability and meaningfulness of invertebrate animal models, in silico analysis and reverse pharmacology are discussed, among other aspects of relevance in today's scenario. Overall, the use of alternative models, including Artemia salina (brine shrimp), Caenorhabditis elegans (roundworm), Danio rerio (zebra fish), Drosophila melanogaster (fruit fly), Galleria mellonella (greater waxmoth) and in silico modelling, increased 909% from 1990 to 2015, as compared to 154% of conventional mammals in the same period. Thus, technological and scientific advancements in the fields of toxicology and drug development seem to have diminished the need for mammalian models. Today, however, mammals still remain critically indispensable to provide - in most cases -reliable data subsidizing and validating translation into the clinical setting344681686FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2011/15984-0; 2013/25080-7310522/2015-

In silico approaches for screening molecular targets in Candida albicans: A proteomic insight into drug discovery and development

Candida species are opportunistic pathogens which can cause conditions ranging from simple mucocutaneous infections to fungemia and death in immunosuppressed and hospitalized patients. Candida albicans is considered to be the species mostly associated with fungal infections in humans and, therefore, the mostly studied yeast. This microorganism has survival and virulence factors which, allied to a decreased host immunity response, make infection more difficult to control. Today, the current limited antifungal arsenal and a dramatic increase in fungal resistance have driven the need for the synthesis of drugs with novel mechanisms of action. However, the development of a new drug from discovery to marketing takes a long time and is highly costly. The objective of this review is to show that with advances in biotechnology and biofinformatics, in silico tools such as molecular docking can optimize such a timeline and reduce costs, while contributing to the design and development of targeted drugs. Here we highlight the most promising protein targets in Candida albicans for the development of drugs with new mechanisms of action8426469CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ141129/2017-

Unexplored endemic fruit species from Brazil: antibiofilm properties, insights into mode of action, and systemic toxicity of four Eugenia spp

CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOBrazilian endemic fruit species have aroused attention due to their highly valuable, yet unexplored, agroindustrial, food and therapeutic potential. Herein, we describe the antifungal activity of four Eugenia spp. against Candida albicans biofilms, and further demonstrate insights into their potential mode(s) of action and toxicity in vitro and in vivo. Extracts from different parts (seeds, pulps, leaves) of E. leitonii (EL), E. brasiliensis (EB), E. myrcianthes (EM) and E. involucrata (EI) were obtained (S23 degrees 23',W45 degrees 39') and chemically characterized by GC/MS. The active extracts were tested against C albicans biofilm viability and architecture, as well as mode of action, and toxicology using RAW 264.7 macrophages and Galleria mellonella larvae. The MIC values ranged from 15.62 to > 2000 mu g/mL. The most active extracts were EL (seed, 15.62 mu g/mL) and EB (leaf and seeds, 31.25 and 15.62 mu g/mL, respectively). Treatment with these extracts at 10xMIC reduced biofilm viability by 54-55% (P 0.05) and G. mellonella larvae, with mean in vivo LD50 of 1500 mg/kg (EL, seeds); 2500 mg/kg (EB, seeds); and 1250 mg/kg (EB, leaf). The phenolic compounds epicatechin and gallic acid were the major constituents in the extracts. Our findings may open avenues for the application of these yet unexplored native fruits in the food and pharmaceutical industry. (C) 2017 Elsevier Ltd. All rights reserved.Brazilian endemic fruit species have aroused attention due to their highly valuable, yet unexplored, agroindustrial, food and therapeutic potential. Herein, we describe the antifungal activity of four Eugenia spp. against Candida albicans biofilms, and further demonstrate insights into their potential mode(s) of action and toxicity in vitro and in vivo. Extracts from different parts (seeds, pulps, leaves) of E. leitonii (EL), E. brasiliensis (EB), E. myrcianthes (EM) and E. involucrata (EI) were obtained (S23 degrees 23',W45 degrees 39') and chemically characterized by GC/MS. The active extracts were tested against C albicans biofilm viability and architecture, as well as mode of action, and toxicology using RAW 264.7 macrophages and Galleria mellonella larvae. The MIC values ranged from 15.62 to > 2000 mu g/mL. The most active extracts were EL (seed, 15.62 mu g/mL) and EB (leaf and seeds, 31.25 and 15.62 mu g/mL, respectively). Treatment with these extracts at 10xMIC reduced biofilm viability by 54-55% (P 0.05) and G. mellonella larvae, with mean in vivo LD50 of 1500 mg/kg (EL, seeds); 2500 mg/kg (EB, seeds); and 1250 mg/kg (EB, leaf). The phenolic compounds epicatechin and gallic acid were the major constituents in the extracts. Our findings may open avenues for the application of these yet unexplored native fruits in the food and pharmaceutical industry105280287CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO474335/201352011/15984-0; 2011/25080-7; 2013/26251-

Phenolic Profile and the Antioxidant, Anti-Inflammatory, and Antimicrobial Properties of Açaí (<i>Euterpe oleracea</i>) Meal: A Prospective Study

The mechanical extraction of oils from Brazilian açaí (Euterpe oleracea Mart) produces significant amounts of a byproduct known as “meal”, which is frequently discarded in the environment as waste material. Nevertheless, plant byproducts, especially those from oil extraction, may contain residual polyphenols in their composition and be a rich source of natural bioactive compounds. In this study, the phenolic composition and in vitro biological properties of a hydroethanolic açaí meal extract were elucidated. The major compounds tentatively identified in the extract by high-resolution mass spectrometry were anthocyanins, flavones, and flavonoids. Furthermore, rhamnocitrin is reported in an açaí byproduct for the first time. The extract showed reducing power and was effective in scavenging the ABTS radical cation (820.0 µmol Trolox equivalent∙g−1) and peroxyl radical (975.7 µmol Trolox equivalent∙g−1). NF-κB activation was inhibited at 10 or 100 µg∙mL−1 and TNF-α levels were reduced at 100 µg∙mL−1. However, the antibacterial effects against ESKAPE pathogens was not promising due to the high concentration needed (1250 or 2500 µg∙mL−1). These findings can be related to the diverse polyphenol-rich extract composition. To conclude, the polyphenol-rich extract obtained from açaí meal showed relevant biological activities that may have great applicability in the food and nutraceutical industries

Environmental isolation, biochemical identification, and antifungal drug susceptibility of Cryptococcus species

Introduction The incidence of opportunistic fungal infections has increased in recent years and is considered an important public health problem. Among systemic and opportunistic mycoses, cryptococcosis is distinguished by its clinical importance due to the increased risk of infection in individuals infected by human immunodeficiency virus. Methods To determine the occurrence of pathogenic Cryptococcus in pigeon excrement in the City of Araraquara, samples were collected from nine environments, including state and municipal schools, abandoned buildings, parks, and a hospital. The isolates were identified using classical tests, and susceptibility testing for the antifungal drugs (fluconazole, itraconazole, voriconazole, and amphotericin B) independently was also performed. After collection, the excrement samples were plated on Niger agar and incubated at room temperature. Results A total of 87 bird dropping samples were collected, and 66.6% were positive for the genus Cryptococcus. The following species were identified: Cryptococcus neoformans (17.2%), Cryptococcus gattii (5.2%), Cryptococcus ater (3.5%), Cryptococcus laurentti (1.7%), and Cryptococcus luteolus (1.7%). A total of 70.7% of the isolates were not identified to the species level and are referred to as Cryptococcus spp. throughout the manuscript. Conclusions Although none of the isolates demonstrated resistance to antifungal drugs, the identification of infested areas, the proper control of birds, and the disinfection of these environments are essential for the epidemiological control of cryptococcosis

Highlights in pathogenic fungal biofilms

A wide variety of fungi have demonstrated the ability to colonize surfaces and form biofilms. Most studies on fungal biofilms have focused on Candida albicans and more recently, several authors have reported the involvement of other genera of yeasts and Candida species, as well as of filamentous fungi in the formation of biofilms, including: Cryptococcus neoformans, Cryptococcus gattii, Rhodotorula species, Aspergillus fumigatus, Malassezia pachydermatis, Histoplasma capsulatum, Paracoccidioides brasiliensis, Pneumocystis species, Coccidioides immitis, Fusarium species, Saccharomyces cerevisiae, Trichosporon asahii, Mucorales and Blastoschizomyces. There is a current interest in describing the particular characteristics of the biofilm formation by of these fungi. A major concern is the control of biofilms, requiring knowledge of the biofilm mechanisms. However, our knowledge of these microbial communities is limited, due to the complexity of these systems and metabolic interactions that remain unknown. This mini-review aims to highlight recently discovered fungal biofilms and to compare them with the current knowledge on biofilms.This manuscript is part of the series of works presented at the V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi (Oaxaca, Mexico, 2012). (C) 2013 Revista Iberoamericana de Micologia. Published by Elsevier Espana, S.L. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Early endosome antigen 1 (EEA1) decreases in macrophages infected with Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM) is a chronic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis, endemic in Latin America. P. brasiliensis has been observed in epithelial cells in vivo and in vitro, as well as within the macrophages. The identification of the mechanism by which it survives within the host cell is fertile ground for the discovery of its pathogenesis since this organism has the ability to induce its own endocytosis in epithelial cells and most likely in macrophages. The study of the expression of endocytic proteins pathway and co-localization of microorganisms enable detection of the mechanism by which microorganisms survive within the host cell. The aim of this study was to evaluate the expression of the endocytic protein EEA1 (early endosome antigen 1) in macrophages infected with P. brasiliensis. For detection of EEA1, three different techniques were employed: immunofluorescence, real-time polymerase chain reaction (PCR) and immunoblotting. In the present study, decreased expression of EEA1 as well as the rearrangement of the actin was observed when the fungus was internalized, confirming that the input mechanism of the fungus in macrophages occurs through phagocytosis. © 2013 ISHAM

Antimicrobial activity of nitrochalcone and pentyl caffeate against hospital pathogens results in decreased microbial adhesion and biofilm formation

The present study investigated the antimicrobial, anti-adhesion and anti-biofilm activity of the modified synthetic molecules nitrochalcone (NC-E05) and pentyl caffeate (C5) against microorganisms which have a high incidence in hospital-acquired infections. The compounds were further tested for their preliminary systemic toxicity in vivo. NC-E05 and C5 showed antimicrobial activity, with minimum inhibitory concentrations (MICs) ranging between 15.62 and 31.25g ml(-1). Treatment with NC-E05 and C5 at 1xMIC and/or 10xMIC significantly reduced mono or mixed-species biofilm formation and viability. At MIC/2, the compounds decreased microbial adhesion to HaCaT keratinocytes from 1 to 3h (p<0.0001). In addition, NC-E05 and C5 demonstrated low toxicity in vivo in the Galleria mellonella model at anti-biofilm concentrations. Thus, the chemical modification of these molecules proved to be effective in the proposed anti-biofilm activity, opening opportunities for the development of new antimicrobials352129142CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES74335/2013-5; 132790/2018-1sem informaçã