Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766) / Obtenção e caracterização de proteases aspárticas digestórias do peixe Caranx hippos (Linnaeus, 1766)

This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

Este trabalho objetivou obter proteases aspárticas de interesse industrial e biotecnológico a partir do estômago do peixe xaréu (Caranx hippos). Para isso, foi obtido um extrato bruto do estômago, o qual foi submetido a uma purificação parcial por salting-out onde se obteve o extrato enzimático (EE). As proteases do EE foram caracterizadas físico-quimicamente e através de zimograma. Além disso, o efeito de agentes químicos sobre sua atividade também foi avaliado. Através de salting-out foi possível obter uma purificação de 1,6 vezes com rendimento de 49,4%. Foram observadas duas proteases ácidas presentes no EE através de zimograma. A temperatura ótima e a estabilidade térmica para as proteases ácidas do EE foram de 55 ºC e 45 °C, respectivamente. O pH ótimo e a estabilidade ao pH encontrados para estas enzimas foram o pH 1,5 e 7,0, respectivamente. Observou-se a inibição total da atividade proteolítica ácida do EE na presença de pepstatina A. O ditiotreitol (DTT) e o Ca2+ não promoveram efeito significativo na atividade enzimática. Na presença de metais pesados, como Al3+, Cd2+ e Hg2+, o EE manteve mais de 70% de atividade enzimática do EE. Os resultados mostram que é possível obter, a partir do estômago de C. hippos, proteases aspárticas com alta atividade proteolítica e características que demonstram potencial para aplicações industriais e biotecnológicas.

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications / Estômago de Carangoides bartholomaei (Cuvier, 1833): uma fonte de proteases aspárticas para aplicações industriais e biotecnológicas

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U·mg-¹. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

As vísceras e outros resíduos do processamento de peixes são geralmente descartados pela indústria pesqueira. Esses resíduos podem ser uma fonte de enzimas digestivas com potencial industrial e biotecnológico. Neste estudo, objetivamos a extração, caracterização e aplicação de proteases aspárticas do estômago de Carangoides bartholomaei (Cuvier, 1833). Um extrato bruto do estômago foi obtido e submetido a um processo de purificação parcial, que obteve um Extrato Purificado (EP) com uma atividade proteolítica específica de 54,0 U·mg-¹. Foi obtida uma purificação de 1,9 vezes e um rendimento de 41%. O EP apresenta duas isoformas de proteases ácidas e atividade proteolítica máxima a 45 °C e pH 2,0. A atividade proteolítica do EP foi estável na faixa de pH de 1,5 a 7,0 e temperatura de 25 °C a 50 °C. O EP manteve 35% de sua atividade proteolítica na presença de NaCl a 15% (m/v), mas foi totalmente inibida pela pepstatina A. As proteases ácidas do EP apresentaram alta atividade na presença de metais pesados como o Cd2+, Hg2+, Pb2+, Al3+ e Cu2+. A utilização de EP como aditivo enzimático na extração de colágeno a partir de escamas de tilápia do Nilo dobrou o rendimento do processo. Os resultados indicam um potencial dessas proteases para aplicações industriais e biotecnológicas.

Gingival Crevicular Fluid Zinc- and Aspartyl-Binding Protease Profile of Individuals with Moderate/Severe Atopic Dermatitis.

Atopic dermatitis (AD) is a protease-modulated chronic disorder with heterogenous clinical manifestations which may lead to an imprecise diagnosis. To date, there are no diagnostic protease tests for AD. We explored the gingival crevicular fluid (GCF) protease profile of individuals with moderate/severe AD compared to healthy controls. An exploratory case-control study was conducted. AD patients (n = 23) and controls (n = 21) were enrolled at the International Center for Clinical Studies, Santiago, Chile. Complete dermatological and periodontal evaluations (involving the collection of GCF samples) were made. The levels of 35 proteases were analyzed using a human protease antibody array in matching AD patients (n = 6) and controls (n = 6) with healthy periodontium. The GCF levels of zinc-binding ADAM8, ADAM9, MMP8, Neprilysin/CD10, aspartyl-binding Cathepsin E, serin-binding Protein convertase9, and uPA/Urokinase proteases were lower in moderate/severe AD patients compared to controls (p < 0.05). No inter-group differences in the levels of the other 28 proteases were found. MMP8, Cathepsin E, and ADAM9 were the biomarkers with the highest sensitivity and specificity regarding the detection of AD (p < 0.05). The area under receiver operating characteristic (ROC) curve for MMP8 was 0.83 and MMP8 + ADAMP9 was 0.90, with no significant differences (p = 0.132). A combined model of MMP8, Cathepsin E, and ADAM9 was not considered since it did not converge. Then, levels of MMP8 in GCF were determined using a multiplex bead immunoassay in 23 subjects with AD and 21 healthy subjects. Lower levels of MMP8 in the GCF from the AD group versus healthy group (p = 0.029) were found. This difference remained significant after adjustment by periodontitis (p = 0.042). MMP8 revealed the diagnostic potential to identify AD patients versus healthy controls, (ROC area = 0.672, p < 0.05). In conclusion, differences in the protease profile between AD and control patients were associated with MMP8, Cathepsin E, and ADAM9. Based on the multiplex assay results, MMP8 was lower in AD patients than controls, suggesting that MMP8 may be a diagnostic biomarker candidate.

Trypanosoma cruzi Presenilin-Like Transmembrane Aspartyl Protease: Characterization and Cellular Localization.

The increasing detection of infections of Trypanosoma cruzi, the etiological agent of Chagas disease, in non-endemic regions beyond Latin America has risen to be a major public health issue. With an impact in the millions of people, current treatments rely on antiquated drugs that produce severe side effects and are considered nearly ineffective for the chronic phase. The minimal progress in the development of new drugs highlights the need for advances in basic research on crucial biochemical pathways in T. cruzi to identify new targets. Here, we report on the T. cruzi presenilin-like transmembrane aspartyl enzyme, a protease of the aspartic class in a unique phylogenetic subgroup with T. vivax separate from protozoans. Computational analyses suggest it contains nine transmembrane domains and an active site with the characteristic PALP motif of the A22 family. Multiple linear B-cell epitopes were identified by SPOT-synthesis analysis with Chagasic patient sera. Two were chosen to generate rabbit antisera, whose signal was primarily localized to the flagellar pocket, intracellular vesicles, and endoplasmic reticulum in parasites by whole-cell immunofluorescence. The results suggest that the parasitic presenilin-like enzyme could have a role in the secretory pathway and serve as a target for the generation of new therapeutics specific to the T. cruzi.

Phenotypical characterization and molecular identification of clinical isolates of Candida tropicalis / Caracterización fenotípica e identificación molecular de aislamientos clínicos de Candida tropicalis

Background. Candida tropicalis is an increasingly important human pathogen which usually affects neutropenic oncology patients with common hematogenous seeding to peripheral organs and high mortality rates. Candida pathogenicity is facilitated by several virulence attributes, including secretion of hydrolytic enzymes; however, little is known regarding the C. tropicalis ability to secrete them and their role in the disease. Aims. To confirm by molecular means the identification of 187 clinical isolates (127 from blood, 52 from urine, and 8 from diverse clinical origins) phenotypically identified as C. tropicalis, and to investigate their in vitro aspartyl proteinase, phospholipase, esterase, hemolysin, DNase and coagulase activities. Methods. The molecular confirmation was performed by ITS sequencing, and the enzymatic determinations were conducted using plate assays with specific substrates, with the exception of coagulase, which was determined by the classical tube test. Results. The majority of the strains exhibited a very strong or strong activity of aspartyl proteinase, phospholipase and esterase. A 4.7% of the bloodstream isolates were hemolysin producers, and all were negative for the coagulase and DNase assays. Conclusions. Very strong activities of aspartyl proteinase, phospholipase and esterase profiles were detected, and a statistical association between phospholipase production and blood and urine isolates was found (AU)

Antecedentes. Candida tropicalis es un patógeno del ser humano cada vez más importante que afecta especialmente a pacientes oncológicos neutropénicos, en los cuales es frecuente la diseminación hematógena del microorganismo a órganos periféricos, lo que conlleva elevadas tasas de mortalidad. La patogenicidad de Candida es facilitada por diversos factores de virulencia, incluyendo la secreción de enzimas hidrolíticas; sin embargo, poco se sabe respecto a la habilidad de C. tropicalis para su secreción, así como el papel que desempeña en la enfermedad. Objetivos. Confirmar por un método molecular la identidad de 187 aislamientos clínicos (127 de sangre, 52 de orina y 8 de orígenes diversos) fenotípicamente identificados como C. tropicalis y estudiar la actividad in vitro de las enzimas proteinasa aspártica, fosfolipasa, esterasa, hemolisina, DNasa y coagulasa. Métodos. La confirmación molecular se llevó a cabo mediante secuenciación del ITS y las determinaciones enzimáticas se llevaron a cabo mediante ensayos en placa con sustratos específicos, a excepción de la coagulasa, que se determinó mediante la clásica prueba en tubo. Resultados. La mayoría de los aislamientos analizados mostraron un perfil de actividad muy fuerte o fuerte de proteinasa aspártica, fosfolipasa y esterasa. El 4,7% de las cepas sanguíneas fue productora de hemolisinas y todas fueron negativas para coagulasa y DNasa. Conclusiones. Se detectaron perfiles con una actividad proteinasa aspártica, fosfolipasa y esterasa muy fuerte entre los aislamientos clínicos analizados, así como también se encontró asociación estadística entre la producción de fosfolipasa y aquellos aislamientos obtenidos de sangre y orina (AU)

Aspartic Protease Zymography Case Study: Detection of Fungal Acid Proteases by Zymography.

This chapter describes a method for the production and characterization of fungal acid proteases. Protease production is induced by growth on BSA media over a pH gradient and protein levels are monitored over time with the Bradford assay. Once protein is depleted, the media is purified and proteases are characterized by gelatin zymography using acrylamide and buffers at near-neutral pH. Maintaining pH levels below those found in traditional zymographic systems avoids the potential loss of activity that may occur in aspartic proteases under alkaline conditions.

Detection of Aspartic Proteinase Activities Using Gel Zymography.

Gel zymography is a two-stage process where the proteins from the test sample are first separated by electrophoresis followed by the detection of the activity of hydrolytic enzymes. Many zymography procedures use sodium dodecyl sulfate (SDS) polyacrylamide gels copolymerized with an appropriate substrate. The procedure described here uses native polyacrylamide gel electrophoresis (PAGE) in the absence of both SDS and substrate. In order to visualize aspartic proteinase activity, the gel is impregnated in bovine hemoglobin at pH 3.0 for 15 min after the electrophoresis procedure. Subsequently, the gel is incubated in a humid container in the absence of hemoglobin for 1 h at 37 °C. At the end, the gel is stained with amido black and destained. Clear areas against a dark background corresponding to aspartic proteinase activities can be detected.

Compartmentalized expression patterns of pancreatic- and gastric-related genes in the alimentary canal of the ascidian Ciona intestinalis: evolutionary insights into the functional regionality of the gastrointestinal tract in Olfactores.

Many heterotrophic animals have a one-way alimentary canal that is essential for their nutrition and sequential steps of the digestive system, namely ingestion, digestion, absorption and elimination, are widely shared among bilaterians. Morphological, functional and molecular knowledge of the alimentary canal has been obtained in particular from mammalian research but the shared features and evolution of these aspects of the highly diverged alimentary canal in the animal kingdom are still unclear. We therefore investigate spatial gene expression patterns of pancreatic- and gastric-related molecules of ascidians (a sister group of vertebrates) with special reference to the functional regionality of the gastrointestinal tract. Genome-wide surveys of ascidian homologs to mammalian exocrine digestive enzyme genes revealed that pancreatic enzymes, namely alpha-amylase, lipase, phospholipase A2, trypsin, chymotrypsin and carboxypeptidase, exist in the ascidian genome. However, an ascidian homolog of the mammalian gastric enzyme pepsin has not been identified, although molecules resembling cathepsin D, a pepsin relative, are indeed present. Spatial expression analyses in the ascidian Ciona intestinalis, by means of whole-mount in situ hybridization, have elucidated that the expression of Ciona homologs of pancreatic- and gastric-related exocrine enzyme genes and of their transcriptional regulator genes is restricted to the Ciona stomach. Furthermore, the expression of these genes is localized to specific regions of the stomach epithelium according to their regionality in the vertebrate digestive system. The compartmentalized expression patterns of Ciona homologs imply primitive and/or ancestral aspects of molecular, functional and morphological bases among Olfactores.

Usefulness of the Non-conventional Caenorhabditis elegans Model to Assess Candida Virulence.

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.

The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors.

Particular peptides generated from the vicilin-class(7S) globulin of the cocoa beans by acid-induced proteolysis during cocoa fermentation are essential precursors of the cocoa-specific aroma notes. As revealed by in vitro studies, the formation of the cocoa-specific aroma precursors depends on the particular cleavage specificity of the cocoa aspartic protease, which cannot be substituted by pepsin. Therefore, we have investigated the effects of aspartic protease inhibitors on both enzymes and comparatively studied their cleavage specificities using different protein substrates and MALDI-TOF mass spectrometric analyses of the generated oligopeptides. Three classes of cleavage sites have been identified and characterized: (I) sequences exclusively cleaved by the cocoa enzyme, (II) sequences cleaved by both pepsin and the cocoa enzyme, and (III) those cleaved exclusively by pepsin. In contrast to most aspartic proteases from other origins, basic amino acid residues, particularly lysine, were found to be abundant in the specific cleavage sites of the cocoa enzyme.

Vacuolar proteases from Candida glabrata: acid aspartic protease PrA, neutral serine protease PrB and serine carboxypeptidase CpY. The nitrogen source influences their level of expression / Proteasas vacuolares de Candida glabrata: aspartil proteasa ácida PrA, proteasa neutra PrB y carboxipeptidasa CpY. La fuente de nitrógeno influye en sus niveles de expresión

Background. The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. Aims. The present paper is the first report on proteolytic activity in the C. glabrata vacuole. Methods. Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. Results. Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. Conclusions. The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen (AU)

Antecedentes. La vacuola de Saccharomyces cerevisiae está involucrada activamente en el mecanismo de autofagia, desarrollando una labor importante en la homeostasis, degradación, recambio proteico, desintoxicación y protección de la célula en condiciones de estrés. Por el contrario, las proteasas vacuolares de Candida glabrata aún no han sido estudiadas por completo. Objetivos. El presente trabajo describe por primera vez la actividad proteolítica vacuolar en C. glabrata. Métodos. Los estudios bioquímicos realizados en C. glabrata pusieron de manifiesto la presencia de diferentes actividades proteolíticas: aspartil proteinasa ácida, que actúa sobre sustratos como la albúmina y la hemoglobina ácida desnaturalizada; serín proteasa neutra, con actividad sobre el substrato de tipo colágeno hide powder azure, y serín carboxipeptidasa, que actúa sobre N-benzoil-tyr-pNa. Resultados. La obtención de una fracción subcelular mostró una elevada actividad enzimática específica de las tres proteasas, lo que permitió confirmar su localización vacuolar. Se realizaron análisis de la expresión de los genes CgPEP4 (CgAPR1), CgPRB1 y CgCPY1 (CgPRC1), codificantes de las actividades proteolíticas aspartil proteasa A, proteasa neutra B y carboxipeptidasa Y, respectivamente. Los resultados reflejan una regulación diferencial de la expresión de la proteasa, dependiendo de la fuente de nitrógeno. Conclusiones. Las proteasas codificadas por los genes CgPEP4, CgPRB1 y CgCPY1 podrían participar en el proceso de autofagia y supervivencia de este patógeno oportunista (AU)

Reliability of the agar based method to assess the production of degradative enzymes in clinical isolates of Candida albicans.

The aim of this study was to establish a reproducible protocol using the methodology of hyaline zones around the colonies on specific agar plates for phospholipase and proteinase production. This was an in vitro double-blind experiment, in which the dependent variables were the enzymatic activity measurements (Pz) for the production of phospholipase (Pz-ph) and the production of secreted aspartyl proteinases (Pz-sap). Three independent variables give rise to different measurement protocols. All measurements were carried out at two different moments by four examiners (E1, E2, E3, and E4). The minimum sample size was 30 Candida albicans clinical isolates. Specific agar plates for phospholipase and SAPs production were prepared according the literature. The intra-and inter-examiner reproducibility for each protocol was estimated using the Intraclass Correlation Coefficient (ICC) and its confidence interval (95% CI). Based on the results obtained for both phospholipase and SAPs, there appears to be no consensus on the protocol chosen for each particular examiner. Measuring the colonies in triplicate may be the main factor associated with the increase in measurement accuracy and should therefore take precedence over measuring only one colony. When only one examiner is responsible for taking measurements, a standard protocol should be put in place and the statistical calibration of this researcher should be done prior to data collection. However, if two or more researchers are involved in the assessment of agar plates, our results suggest that the protocols using software to undertake plate reading is preferred.

iTRAQ-based quantitative proteomics analysis of rice leaves infected by Rice stripe virus reveals several proteins involved in symptom formation.

BACKGROUND: Rice plants infected by Rice stripe virus (RSV) usually leads to chlorosis and death of newly emerged leaves. However, the mechanism of RSV-induced these symptoms was not clear. METHODS: We used an iTRAQ approach for a quantitative proteomics comparison of non-infected and infected rice leaves. RT-qPCR and Northern blot analyses were performed for assessing the transcription of candidate genes. RESULTS: As a whole, 681 (65.8% downregulated, 34.2% upregulated infected vs. non-infected) differentially accumulated proteins were identified. A bioinformatics analysis indicated that ten of these regulated proteins are involved in chlorophyll biosynthesis and three in cell death processes. Subsequent RT-qPCR results showed that downregulation of magnesium chelatase was due to reduced expression levels of the genes encoding subunits CHLI and CHLD, which resulted in chlorophyll reduction involved in leaf chlorosis. Three aspartic proteases expressed higher in RSV-infected leaves than those in the control leaves, which were also implicated in RSV-induced cell death. Northern blot analyses of CHLI and p0026h03.19 confirmed the RT-qPCR results. CONCLUSIONS: The magnesium chelatase and aspartic proteases may be associated with RSV-induced leaf chlorosis and cell death, respectively. The findings may yield new insights into mechanisms underlying rice stripe disease symptom formation.

Phospholipase and Aspartyl Proteinase Activities of Candida Species Causing Vulvovaginal Candidiasis in Patients with Type 2 Diabetes Mellitus.

Few research had investigated the secretion of phospholipase and aspartyl proteinase from Candida spp. causing infection in females with type 2 diabetes mellitus. This research aimed to investigate the prevalence of vulvovaginal candidiasis (VVC) in diabetic versus non-diabetic women and compare the ability of identified Candida isolates to secrete phospholipases and aspartyl proteinases with characterization of their genetic profile. The study included 80 females with type 2 diabetes mellitus and 100 non-diabetic females within the child-bearing period. Candida strains were isolated and identified by conventional microbiological methods and by API Candida. The isolates were screened for their extracellular phospholipase and proteinase activities by culturing them on egg yolk and bovine serum albumin media, respectively. Detection of aspartyl proteinase genes (SAP1 to SAP8) and phospholipase genes (PLB1, PLB2) were performed by multiplex polymerase chain reaction. Our results indicated that vaginal candidiasis was significantly higher among the diabetic group versus nondiabetic group (50% versus 20%, respectively) (p = 0.004). C. albicans was the most prevalent species followed by C. glabrata in both groups. No significant association between diabetes mellitus and phospholipase activities was detected (p = 0.262), whereas high significant proteinase activities exhibited by Candida isolated from diabetic females were found (82.5%) (p = 0.000). Non-significant associations between any of the tested proteinase or phospholipase genes and diabetes mellitus were detected (p > 0.05). In conclusion, it is noticed that the incidence of C. glabrata causing VVC is increased. The higher prevalence of vaginal candidiasis among diabetics could be related to the increased aspartyl proteinase production in this group of patients.

Enzymatic and hemolytic activity in different Candida species / La actividad enzimática y hemolítica en diferentes especies de Candida

Background: Candida species, in conditions of microbiota imbalance or decreased immune defenses, may be one of the main human fungal pathogens. Virulence factors constitute the mechanisms used by the fungus to avoid host defenses. Aims: This study aimed to investigate the in vitro production of virulence factors, such as hemolytic activity, and deoxyribonuclease (DNase), proteinase, and phospholipase activities in Candida spp. Methods: Fifty clinical isolates were analyzed for virulence factors: Candida albicans (15), Candida tropicalis (15), Candida parapsilosis (10), Candida glabrata (5), and Candida krusei (5). Hemolytic activity was determined in Sabouraud dextrose agar plates containing 3% glucose and 7% sheep red cells. Culture media containing, respectively, agar-base DNA, egg yolk, and bovine albumin were used to determine DNase, phospholipase and proteinase activities, respectively. Results: Forty-eight (96%) of 50 isolates showed hemolytic activity, with 10 (20%) positive for DNase, 19 (38%) for proteinase, and 16 (32%) for phospholipase. Statistically significant differences were observed between species for phospholipase (p < 0.0001) and proteinase (p < 0.05) production. Conclusions: It is concluded that all species had hemolytic activity. DNase activity was detected in all species except in C. glabrata; proteinase activity was detected in C. albicans, C. tropicalis, and C. parapsilosis; and phospholipase activity was observed in C. albicans and C. tropicalis (AU)

Antecedentes: Las levaduras del género Candida, en condiciones de desequilibrio de la microbiota o de disminución de las defensas inmunológicas, pueden ser uno de los principales patógenos fúngicos del hombre. Los factores de virulencia constituyen los mecanismos utilizados por el hongo para evadir las defensas del huésped. Objetivos: Este estudio tiene como objetivo investigar la producción in vitro de algunos factores de virulencia, como la actividad hemolítica, y las actividades desoxirribonucleasa (DNasa), proteinasa y fosfolipasa en Candidaspp. Métodos: Se analizaron 50 aislamientos clínicos: Candida albicans (15), Candida tropicalis (15), Candida parapsilosis (10), Candida glabrata (5), y Candida krusei (5). La actividad hemolítica fue determinada en placas de agar glucosado de Sabouraud, con glucosa al 3% y un 7% de hematíes de oveja. Los medios de cultivo de agar-ADN, yema de huevo y albúmina bovina fueron utilizados para determinar las actividades DNasa, fosfolipasa y proteinasa, respectivamente. Resultados: De los 50 aislamientos, 48 (96%) presentaron actividad hemolítica, 10 (20%) fueron positivos para DNasa, 19 (38%) para proteinasa y 16 (32%) para fosfolipasa. Se observaron diferencias estadísticamente significativas entre las especies para las actividades fosfolipasa (p < 0,0001) y proteasa (p < 0,05). Conclusiones: Se concluye que todas las especies estudiadas poseen actividad hemolítica. La actividad DNasa fue detectada en todas las especies, excepto en Candida glabrata; la actividad proteinasa fue detectada en C. albicans, C. tropicalis y C. parapsilosis, y la actividad fosfolipasa se observó en C. albicans y C. tropicalis (AU)

Quantitative analysis of isomeric (l-α-, l-ß-, D-α-, D-ß-) aspartyl residues in proteins from elderly donors.

Homochirality is essential for life. For a long time, it was considered that d-amino acids were excluded from living systems. In the past 30 years, however, d-amino acids have been found in living organisms in the form of free amino acids, peptides and proteins, owing to advances in the analysis of optical isomers of amino acids. Free D-amino acids and D-amino-acid-containing peptides have been shown to have important physiological functions. The amount of D-aspartate (Asp) residues in protein spontaneously increases in metabolically inert tissues such as the eye and brain during aging, and may be related to cataract formation and the development of Alzheimer disease, suggesting that D-Asp might be a molecular marker of aging and age-related disorders. The presence of D-Asp in living organisms is thought to result from the isomerization of L-Asp residues in some proteins. Furthermore, the isomerization of Asp does not occur uniformly but only at specific sites. Therefore, it is necessary to determine the sites of isomeric Asp in these proteins in order to elucidate the mechanism of spontaneous Asp isomerization during aging. Herein, we summarize the localization and mechanism of D-amino acids in proteins of living tissues, and the effects of D-amino acid formation in proteins. Furthermore, we describe methods for the analysis of protein-bound D-amino acids including a conventional enantioseparation method based on HPLC and a new convenient method based on LC-MS that can identify the specific sites of D-Asp in proteins.

Plasma proteins in the acquired denture pellicle enhance substrate surface free energy and Candida albicans phospholipase and proteinase activities.

AIM: The objective of the present study was to determine if blood plasma proteins could change the proteome of the acquired denture pellicle by label-free quantitative proteomics. As pellicle proteome modulates the interaction between substrates and Candida cells, we investigated its effect on the surface free energy (SFE) of the coated resin and on Candida albicans phospholipase and aspartyl proteinase activities. METHODS: Poly(methylmethacrylate) discs were exposed to saliva (control) or saliva enriched with blood plasma (experimental group). The pellicle proteome was analyzed by mass spectrometry coupled with liquid chromatography. SFE was determined by acid-base technique. After biofilm formation, phospholipase and proteinase activities were determined accordingly to classic plate methods. Data were analyzed by two-way anova and Tukey test (P < 0.05). RESULTS: α-Amylase, cystatins, mucins, and host-immune system proteins were the main proteins identified in the control group. Fibrinogen and albumin were observed only in the experimental group. Coated discs of the experimental group presented an increased SFE (P < 0.05). For both enzymes tested, the experimental group showed higher proteolytic activity (P < 0.001). CONCLUSION: Blood plasma changes the proteome of the acquired denture pellicle, increasing surface free energy and the activity of Candida albicans phospholipase and aspartyl proteinase.

Effect of daily use of an enzymatic denture cleanser on Candida albicans biofilms formed on polyamide and poly(methyl methacrylate) resins: an in vitro study.

STATEMENT OF PROBLEM: Candida biofilms on denture surfaces are substantially reduced after a single immersion in denture cleanser. However, whether this effect is maintained when dentures are immersed in cleanser daily is unclear. PURPOSE: The purpose of this study was to evaluate the effect of the daily use of enzymatic cleanser on Candida albicans biofilms on denture base materials. MATERIAL AND METHODS: The surfaces of polyamide and poly(methyl methacrylate) resin specimens (n=54) were standardized and divided into 12 groups (n=9 per group), according to study factors (material type, treatment type, and periods of treatment). Candida albicans biofilms were allowed to form over 72 hours, after which the specimens were treated with enzymatic cleanser once daily for 1, 4, or 7 days. Thereafter, residual biofilm was ultrasonically removed and analyzed for viable cells (colony forming units/mm(2)) and enzymatic activity (phospholipase, aspartyl-protease, and hemolysin). Factors that interfered with the response variables were analyzed by 3-way ANOVA with the Holm-Sidak multiple comparison method (α=.05). RESULTS: Polyamide resin presented more viable cells of Candida albicans (P<.001) for both the evaluated treatment types and periods. Although enzymatic cleansing significantly (P<.001) reduced viable cells, daily use did not maintain this reduction (P<.001). Phospholipase activity significantly increased with time (P<.001) for both materials and treatments. However, poly(methyl methacrylate) based resin (P<.001) and enzymatic cleansing treatment (P<.001) contributed to lower phospholipase activity. Aspartyl-protease and hemolysin activities were not influenced by study factors (P>.05). CONCLUSIONS: Although daily use of an enzymatic cleanser reduced the number of viable cells and phospholipase activity, this treatment was not effective against residual biofilm over time.

Enzymatic activity profile of a Brazilian culture collection of Candida albicans isolated from diabetics and non-diabetics with oral candidiasis.

The secretion of hydrolytic enzymes is a fundamental virulence factor of Candida albicans to develop disease. The objective of this study was to characterise the virulence of 148 clinical isolates of C. albicans from oral candidiasis by assessing the expression of phospholipase (PL) and secreted aspartyl proteinase (SAP). Isolates were obtained from healthy subjects (HS) and diabetics (DOC) and non-diabetics with oral candidiasis (NDOC). An aliquot (5 µl) of each cell suspension was inoculated on PL and SAP agar plates and incubated. Enzymes secretion was detected by the formation of an opaque halo around the colonies and enzymatic activity (PZ) was determined by the ratio between colony diameter and colony diameter plus the halo zone. Statistical comparisons were made by a one-way anova followed by Tukey's post hoc test (α = 0.05). The clinical sources of C. albicans had significant effect (P < 0.001) on the PZ values of both enzymes. For PL, clinical isolates from NDOC and DOC had highest enzymatic activity than those from HS (P < 0.05), with no significant differences between them (P = 0.506). For SAP, C. albicans from NDOC showed the lower enzymatic activity (P < 0.001). There were no significant differences between isolates from HS and DOC (P = 0.7051). C. albicans isolates from NDOC and DOC patients showed an increased production of PL.

Aspartyl proteinase, phospholipase, esterase and hemolysin activities of clinical isolates of the Candida parapsilosis species complex.

Candida parapsilosis is considered as an important emerging fungal pathogen and was recently found to be a complex that include three species, i.e., Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis. The aim of this study was to determine the in vitro aspartyl proteinase, phospholipase, esterase and hemolysin activities of 65 clinical isolates of the C. parapsilosis complex, which had been previously identified by RFLP-BanI analysis. Of the enzymes evaluated, aspartyl proteinase was the least produced by the C. parapsilosis species complex. Phospholipase and esterase were strongly expressed by C. orthopsilosis (67% of isolates), while 10% and 13% of C. parapsilosis sensu stricto isolates were strong producers, respectively, of these two enzymes. In contrast, high production of both enzymes was not detected in C. metapsilosis. Hemolysin activity was significantly more abundant in C. orthopsilosis (87%) than C. parapsilosis sensu stricto (67%). Overall, C. orthopsilosis isolates were statistically associated with the production of hemolysins (P= 0.048) and phospholipases (P< 0.0001) compared to isolates of C. parapsilosis sensu stricto or C. metapsilosis. Furthermore, a statistical association was found between isolates recovered from blood and phospholipase production (P= 0.017). The distribution of isolates obtained from blood was 30% of C. parapsilosis sensu stricto, 67% of C. orthopsilosis and 20% of C. metapsilosis.