Transcriptome profile of the response of paracoccidioides spp. to a camphene thiosemicarbazide derivative

ABSTARCT: Paracoccidioidomycosis (PCM) is a systemic granulomatous human mycosis caused by fungi of the genus Paracoccidioides, which is geographically restricted to Latin America. Inhalation of spores, the infectious particles of the fungus, is a common route of infection. The PCM treatment of choice is azoles such as itraconazole, but sulfonamides and amphotericin B are used in some cases despite their toxicity to mammalian cells. The current availability of treatments highlights the need to identify and characterize novel targets for antifungal treatment of PCM as well as the need to search for new antifungal compounds obtained from natural sources or by chemical synthesis. To this end, we evaluated the antifungal activity of a camphene thiosemicarbazide derivative (TSC-C) compound on Paracoccidioides yeast. To determine the response of Paracoccidioides spp. to TSC-C, we analyzed the transcriptional profile of the fungus after 8 h of contact with the compound. The results demonstrate that Paracoccidioides lutzii induced the expression of genes related to metabolism; cell cycle and DNA processing; biogenesis of cellular components; cell transduction/signal; cell rescue, defense and virulence; cellular transport, transport facilities and transport routes; energy; protein synthesis; protein fate; transcription; and other proteins without classification. Additionally, we observed intensely inhibited genes related to protein synthesis. Analysis by fluorescence microscopy and flow cytometry revealed that the compound induced the production of reactive oxygen species. Using an isolate with down-regulated SOD1 gene expression (SOD1-aRNA), we sought to determine the function of this gene in the defense of Paracoccidioides yeast cells against the compound. Mutant cells were more susceptible to TSC-C, demonstrating the importance of this gene in response to the compound. The results presented herein suggest that TSC-C is a promising candidate for PCM treatment

Candidiasis oral en neonatos: Caracterización de aislamientos de cándida por análisis del ADN

In a 1-year period, from 04-21-94 to 04-21-95, in the newbom ward of the Hospital General de Medellín, the incidence of oral candidiasis was 2.4% (21 patients). From each patient, a clínical isolation of Candida was obtained. Candida albicans was identified from 20 patients (95.2%) and Candida parapsilosis from one patient. These isolates were studied by restriction fragment length polymorphlsm analísis using.the restriction enzymes Eco RI, and Msp 1. The patterns of restrictlOn fragment lenght polymorphisms of the DNA extracted from the C. albicans isolates were different from each other, except for two pairs of isolates. These two pairs shared the same restriction profile beetwen them, and were epidemiologically connected. This suggested that nosocomial patient to patient cross infection with C. albicans ocurred twice. The C. parapsilosis isolated was found to have a different restriction profile from those of C. albicans Isolations using both enzymes.Se realizó un estudio descriptivo, prospectivo, en el Servicio de Recién Nacidos del Hospital General de Medellín durante el período comprendido entre el21 de abril de 1994 y el21 de abril de 1995. Se encontró que la incidencia de candidiasis oral clínica fue del 2.4% (21 pacientes). De las 21 cepas aisladas, 20 (95.2%) se clasificaron como Candida albicans y una cepa se identificó como Candida parapsilosis. La prueba de polimorfismo en la longitud de los fragmentos de restricción con la enzima Eco RI, así como con Msp 1, de cada uno de los 20 aislamientos de C. albicans mostró patrones de bandas diferentes para cada aislamiento, excepto en dos pares, los cuales compartían entre sí el mismo perfil de restricción. Esto indica que hubo transmisión nosocomial de C. albicans de un paciente a otro en dos ocasiones. Cada pareja de pacientes a los que pertenecían los aislamientos que mostraron patrones idénticos estaban relacionados epidemiológicamente en tiempo y espacio. El aislamiento de C. parapsilosis mostró un patrón de bandas diferente a los obtenidos de los aislamientos de C. albicans con ambas endonucleasas

Capacity of Histoplasma capsulatum to Survive the Composting Process

Histoplasma capsulatum (H. capsulatum) is a thermal-dimorphic fungus, the causal agent of histoplasmosis. Its presence in the environment is related with chicken manure due to their high nitrogen and phosphorus content. In Colombia, chicken manure is the most used raw material in the composting process; however, there is no information about the capacity of H. capsulatum to survive and remain viable in a composted organic fertilizer. To address this question, this study shows three assays based on microbiological culture and the Hc100 nested PCR. First, a composting reactor system was designed to transform organic material under laboratory conditions, and the raw material was inoculated with the fungus. From these reactors, the fungus was not isolated, but its DNA was detected. In the second assay, samples from factories where the DNA of the fungus was previously detected by PCR were analyzed. In the raw material samples, 3 colonies of H. capsulatum were isolated and its DNA was detected. However, after the composting process, neither the fungus was recovered by culture nor DNA was detected. In the third assay, sterilized and nonsterilized organic composted samples were inoculated with H. capsulatum and then evaluated monthly during a year. In both types of samples, the fungus DNA was detected by Hc100 nested PCR during the whole year, but the fungus only grew from sterile samples during the first two months evaluated. In general, the results of the assays showed that H. capsulatum is not able to survive a well-done composting process. © 2019 Luisa Fernanda Gómez Londoño et al

Hiperplasia pseudoepiteliomatosa: Carcinoma escamo celular vs paracoccidioidomicosis oral. Un caso con mirada dermatológica.

Paracoccidioidomycosis is an endemic systemic mycosis in Latin America. The most frequent form of appearance involves the lungs, skin and mucosa chronically. In this case presentation at the beginning and for several years of a single oral lesion in the absence of other symptoms points to a malignant neoplasm, especially a squamous cell carcinoma. The differentiation is made through direct examination and histopathological study. However, in this case the direct examination, the histopathological study and the initial and subsequent cultures were not conclusive. The coexistence of both diagnoses is frequent and increases the diagnostic challenge. This case warns of a systematical absence of clinical suspicion that mucocutaneous lesions maybe produced by fungi that cause endemic mycosis as Paracoccidioides spp and the importance of considering this agent among differential diagnoses.Paracoccidioidomicosis es una micosis sistémica endémica en Latinoamérica. La presentación más frecuente compromete los pulmones, la piel y las mucosas crónicamente. En este paciente se presentó inicialmente y por varios años, una lesión única en mucosa oral que en ausencia de otros síntomas hizo que la sospecha inicial fuera una neoplasia maligna, especialmente un carcinoma escamocelular, donde la diferencia se realiza a través del examen directo y estudio histopatológico. No obstante, en este caso el examen directo, el estudio histopatológico y los cultivos iniciales y subsecuentes no fueron concluyentes. El caso que se reporta alerta sobre la ausencia de sospecha clínica de que las lesiones mucocutáneas pueden ser producidas por hongos causantes de micosis endémicas como Paracoccidioides spp y la importancia de considerar este agente entre los diagnósticos diferenciales

Effects of Argentilactone on the Transcriptional Profile, Cell Wall and Oxidative Stress of Paracoccidioides spp.

Paracoccidioides spp., a dimorphic pathogenic fungus, is the etiologic agent of paracoccidioidomycosis (PCM). PCM is an endemic disease that affects at least 10 million people in Latin America, causing severe public health problems. The drugs used against pathogenic fungi have various side effects and limited efficacy; therefore, there is an inevitable and urgent medical need for the development of new antifungal drugs. In the present study, we evaluated the transcriptional profile of Paracoccidioides lutzii exposed to argentilactone, a constituent of the essential oil of Hyptis ovalifolia. A total of 1,058 genes were identified, of which 208 were up-regulated and 850 were down-regulated. Cell rescue, defense and virulence, with a total of 26 genes, was a functional category with a large number of genes induced, including heat shock protein 90 (hsp90), cytochrome c peroxidase (ccp), the hemoglobin ligand RBT5 (rbt5) and superoxide dismutase (sod). Quantitative real-time PCR revealed an increase in the expression level of all of those genes. An enzymatic assay showed a significant increase in SOD activity. The reduced growth of Pbhsp90-aRNA, Pbccp-aRNA, Pbsod-aRNA and Pbrbt5-aRNA isolates in the presence of argentilactone indicates the importance of these genes in the response of Paracoccidioides spp. to argentilactone. The response of the P. lutzii cell wall to argentilactone treatment was also evaluated. The results showed that argentilactone caused a decrease in the levels of polymers in the cell wall. These results suggest that argentilactone is a potential candidate for antifungal therapy

Caracterización fenotípica y genotípica de aislamientos clínicos colombianos de Sporothrix spp.

Introduction: Over a century, Sporothrix schenckii was considered the only species responsible for sporotrichosis, years later in 2007 it became evident that the disease could be caused by different Sporothrix species that differed in both their virulence factors and antifungals susceptibility among them. Objectives: To characterize phenotypical and genotypically 42 Colombian clinical isolates of Sporothrix spp.Material and methods: 42 clinical isolates were characterized using phenotypic methods that include: several culture media to determine range of growth at different temperatures, type and distribution of their pigment and the texture of the colonies. Additionally, the microscopic morphology was evaluated by microcultures determining the diameter and type of sporulation and the morphology of the conidia. The assimilation of carbohydrates was also used as a physiological trait for species identification. Genotyping of 40 isolates was carried out by partial amplification of the calmodulin gene and sequence analysis.Results: Molecular studies permitted the identification of 32 S. schenckii and 8 S. globosa isolates. The combination of phenotypic and genotypic methods allowed to characterize the species and allowed to construct keys for their recognition based on parameters such as diameter of growth at 25 ºC, 30 ºC, colony texture in PDA (membranous, velvety), and microscopic morphology with predominance of conidia (pigmented triangular, elongated oval globose, subglobose).Conclusions: the confirmation of the phenotypic characteristics and molecular analyzes should be performed in order to identify the Sporothrix species allowing to decide on proper treatment. This is the first phenotypical and genotypical characterization of Colombian Sporothrix spp. clinical isolates.Introducción. Por más de un siglo, Sporothrix schenckii se pensó era la única especie responsable de la esporotricosis, en el 2007 se consideró que puede ser causada por diferentes especies de Sporothrix que difieren en sus factores de virulencia y susceptibilidad a antifúngicos.Objetivos. Caracterizar fenotípica y genotípicamente 42 aislamientos clínicos Colombianos de Sporothrix spp.Materiales y métodos. 42 aislamientos clínicos se caracterizaron usando métodos fenotípicos: varios medios de cultivo para determinar: rango de crecimiento a diferentes temperaturas, tipo y distribución del pigmento y textura de las colonias. Se evaluó la morfología microscópica por microcultivos determinando el diámetro y tipo de esporulación y la morfología de las conidias. La asimilación de carbohidratos se usó como una característica fisiológica para la identificación de especies. La genotipificación de 40 aislamientos se llevó a cabo mediante la amplificación parcial del gen de calmodulina y secuenciación.Resultados. Mediante estudios moleculares se identificaron los aislamientos: 32 S. schenckii y 8 S. globosa. La combinación de métodos fenotípicos y genotípicos permitió caracterizar las especies y construir claves para su reconocimiento basado en parámetros tales como el diámetro de crecimiento a 25 ºC, 30 ºC, textura de las colonias en PDA (membranosa, aterciopelada), y la morfología microscópica con predominio de conidias (triangulares pigmentadas, ovales globosas elongadas, subglobosas). Conclusiones. La caracterización fenotípica y análisis moleculares deben realizarse para poder identificar las especies de Sporothrix y de esta forma decidir el tratamiento indicado. Esta es la primera caracterización fenotípica y genotípica de aislamientos clínicos Colombianos de Sporothrix spp

Susceptibility of <i>Paracoccidioides brasiliensis</i> yeast cells exposed to argentilactone.

<p>Samples containing 10⁶, 10⁵ and 10⁴<i>Pbsod</i>-aRNA, <i>Pbhsp90</i>-aRNA <b>(A)</b>, <i>Pbccp</i>-aRNA and <i>Pbrbt5</i>-aRNA <b>(B)</b> yeast cells were spotted in solid Fava Netto’s supplemented with argentilactone at the concentrations of 4.5, 9, 18 and 36 μg/mL. Control cells, wild type (WT) and empty vector (EV) were assayed without argentilactone. The plates were incubated for 7 days at 36°C before photo documentation.</p

Ethanol dosage in <i>Paracoccidioides lutzii</i> under the action of argentilactone.

<p>A total of 10⁶ cells were used for each sample, and the ethanol levels in the presence and absence of argentilactone were quantified using enzymatic detection. The data are expressed as the mean ± standard deviation of biological triplicates of independent experiments. Student's <i>t</i>-test was used. *, significantly different from the carbon condition at a <i>p-</i>value of ≤ 0.05.</p

Effect of argentilactone on the polymer levels of the <i>Paracoccidioides lutzii</i> cell wall.

<p>Fluorescence microscopy showing yeast cells stained by CFW and CR: <b>(A)</b><i>P</i>. <i>lutzii</i> control stained with CR; <b>(B)</b><i>P</i>. <i>lutzii</i> after treatment with argentilactone, stained with CR; <b>(C)</b><i>P</i>. <i>lutzii</i> control stained with CFW; <b>(D)</b><i>P</i>. <i>lutzii</i> after treatment argentilactone, stained with CFW.</p