Extracellular lipids of Candida albicans biofilm induce lipid droplet formation and decreased response to a topoisomerase I inhibitor in dysplastic and neoplastic oral cells

Abstract Objective Some microorganisms, i.e., Candida albicans, have been associated with cancer onset and development, although whether the fungus promotes cancer or whether cancer facilitates the growth of C. albicans is unclear. In this context, microbial-derived molecules can modulate the growth and resistance of cancer cells. This study isolated extracellular lipids (ECL) from a 36-h Candida albicans biofilm incubated with oral dysplastic (DOK) and neoplastic (SCC 25) cells, which were further challenged with the topoisomerase I inhibitor camptothecin (CPT), a lipophilic anti-tumoral molecule. Methodology ECL were extracted from a 36-h Candida albicans biofilm with the methanol/chloroform precipitation method and identified with Nuclear Magnetic Resonance (1H-NMR). The MTT tetrazolium assay measured ECL cytotoxicity in DOK and SCC 25 cells, alamarBlue™ assessed cell metabolism, flow cytometry measured cell cycle, and confocal microscopy determined intracellular features. Results Three major classes of ECL of C. albicans biofilm were found: phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylglycerol (PG). The ECL of C. albicans biofilm had no cytotoxic effect on neither cell after 24 hours, with a tendency to disturb the SCC 25 cell cycle profile (without statistical significance). The ECL-induced intracellular lipid droplet (LD) formation on both cell lines after 72 hours. In this context, ECL enhanced cell metabolism, decreased the response to CPT, and modified intracellular drug distribution. Conclusion The ECL (PI, PC, and PG) of 36-h Candida albicans biofilm directly interacts with dysplastic and neoplastic oral cells, highlighting the relevance of better understanding C. albicans biofilm signaling in the microenvironment of tumor cells.

Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immuno assay in horses from São Paulo State, Brazil / Produção da proteína recombinante EMA-1 e sua aplicação para o diagnóstico baseadono imuno ensaio enzimático de Theileria equi em equinos do Estado de São Paulo, Brasil

The erythrocytic-stage surface protein, Equi Merozoite Antigen 1 (EMA-1), is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding the entire EMA-1 of Theileria equi Jaboticabal strain was cloned and expressed in Escherichia coli as a histidine-tagged protein (His6-EMA1). The expressed EMA-1 reacted with specific antibodies in Western blot and had an apparent molecular mass of 34 kDa which was largely consistent with its theoretical value. The nucleotide sequence of the EMA-1 gene of Jaboticabal strain was comparatively analyzed with other published sequences. The results indicated a high degree of homology with EMA-1 genes of all other strains isolated from various countries. The recombinant purified His6-EMA1 protein was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies anti-T. equi in horses. The ELISA clearly differentiated T. equi-infected from Babesia caballi-infected horse sera or normal horse sera. Field serum samples collected from horses in the State of São Paulo, Southeastern Brazil, were examined for the diagnosis of T. equi infection by ELISA. Of 170 samples analyzed, 95.88 percent (163/170) were positive for T. equi infection. These results suggest that the His6-EMA1 protein expressed in E. coli could be a reliable immunodiagnostic antigen for ELISA test and that T. equi infection is a serious concern in the State of São Paulo, Brazil.

A proteína de superfície eritrocitária, Antígeno 1 do Merozoíta de Theileria equi (EMA-1), é um potencial candidato para o desenvolvimento de antígenos de valor diagnóstico para a piroplasmose equina. Com o objetivo de estabelecer um método de diagnóstico efetivo e prático, o gene EMA-1 da amostra Jaboticabal - SP de T. equi foi clonado e expresso em Escherichia coli contendo uma cauda de poli-histidina (His6-EMA1). O EMA-1 expresso reagiu com anticorpos específicos no Western blot e apresentou peso molecular aparente de 34 kDa, sendo altamente consistente com seu valor teórico. A sequência nucleotídica do gene EMA-1 da amostra Jaboticabal foi analisado comparativamente com outras sequências públicas, e os resultados indicam elevado grau de homologia com amostras de diversos países. A proteína recombinante purificada His6-EMA1 foi testada no ensaio imunoenzimático (ELISA) para a detecção de anticorpos anti-T. equi em equinos. O teste de ELISA diferenciou-se claramente entre soros de equinos infectados por T. equi, soros de animais infectados por Babesia caballi e soro normal de equino. Amostras de soros coletadas de equinos do Estado de São Paulo, sudeste do Brasil, foram examinadas para o diagnóstico da infecção por T. equi pelo ELISA. Das 170 amostras analisadas, 95,88 por cento (163/170) foram positivas para T. equi. Os resultados sugerem que a proteína His6-EMA1 expressa em E. coli pode ser um antígeno confiável para diagnóstico imunológico pelo teste de ELISA, e que T. equi merece grande atenção no Estado de São Paulo.