Characterization of Extracellular Vesicles Secreted by a Clinical Isolate of Naegleria fowleri and Identification of Immunogenic Components within Their Protein Cargo

Abstract: Extracellular vesicles (EVs) are small lipid vesicles released by both prokaryotic and eu-karyotic cells, involved in intercellular communication, immunomodulation and pathogenesis. In this study, we performed a characterization of the EVs produced by trophozoites of a clinical isolate of the free-living amoeba Naegleria fowleri (N. fowleri). Size distribution, zeta potential, protein profile and protease activity were analyzed. Under our incubation conditions, EVs of different sizes were observed, with a predominant population ranging from 206 to 227 nm. SDS-PAGE revealed protein bands of 25 to 260 KDa. The presence of antigenic proteins was confirmed by Western blot, which evidenced the strongest recognition by rat polyclonal antibodies raised against N. fowleri in the region close to 80 KDa and included peptidases, as revealed by zymography. Proteins in the selected immunorecognized bands were further identified using nano-ESI-MS/MS. A preliminary proteomic profile of the EVs identified at least 184 proteins as part of the vesicles’ cargo. Protease activity assays, in combination with the use of inhibitors, revealed the predominance of serine proteases. The present characterization uncovers the complexity of EVs produced by N. fowleri, suggesting their potential relevance in the release of virulence factors involved in pathogenicity. Owing to their cargo’s diversity, further research on EVs could reveal new therapeutic targets or biomarkers for developing rapid and accurate diagnostic tools for lethal infections, such as the one caused by this amoeba.Vicerrectoría de Investigación/[803-C1-061]/VI/Costa RicaVicerrectoría de Investigación/[803-C2-600]/VI/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Providing an in vitro depiction of microglial cells challenged with immunostimulatory extracellular vesicles of Naegleria fowleri

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis, a rapid and acute infection of the central nervous system with a fatal outcome in >97% of cases. Due to the infrequent report of cases and diagnostic gaps that hinder the possibility of recovering clinic isolates, studies related to pathogenesis of the disease are scarce. However, the secretion of cytolytic molecules has been proposed as a factor involved in the progression of the infection. Several of these molecules could be included in extracellular vesicles (EVs), making them potential virulence factors and even modulators of the immune response in this infection. In this work, we evaluated the immunomodulatory effect of EVs secreted by two clinic isolates of Naegleria fowleri using in vitro models. For this purpose, characterization analyses between EVs produced by both isolates were first performed, for subsequent gene transcription analyses post incubation of these vesicles with primary cultures from mouse cell microglia and BV-2 cells. Analyses of morphological changes induced in primary culture microglia cells by the vesicles were also included, as well as the determination of the presence of nucleic acids of N. fowleri in the EV fractions. Results revealed increased expression of NOS, proinflammatory cytokines IL-6, TNF-α, and IL-23, and the regulatory cytokine IL-10 in primary cultures of microglia, as well as increased expression of NOS and IL-13 in BV-2 cells. Morphologic changes from homeostatic microglia, with small cellular body and long processes to a more amoeboid morphology were also observed after the incubation of these cells with EVs. Regarding the presence of nucleic acids, specific Naegleria fowleri DNA that could be amplified using both conventional and qPCR was confirmed in the EV fractions. Altogether, these results confirm the immunomodulatory effects of EVs of Naegleria fowleri over microglial cells and suggest a potential role of these vesicles as biomarkers of primary acute meningoencephalitis.Projects C-2600: “Secreción de vesículas extracelulares por Naegleria fowleri y evaluación de su potencial rol inmunomodulador en un modelo in vitro” and C-1061: “Caracterización de antígenos de excreción/secreción y antígenos somáticos en amebas de vida libre mediante el empleo de anticuerpos policlonales producidos en roedores”, supported by Vicerrectoría de Investigación of the University of Costa RicaOficina de Asuntos Internacionales y Cooperación Externa (OAICE) of Universidad de Costa RicaFundación Carolina (Spain)“Contrato de Formación de Profesorado Universitario” of the Ministry of Science, Culture and Sports (Spain

Angiostrongiliasis abdominal: notas sobre el diagnóstico

Abdominal angiostrongyliasis, caused by Angiostrongylus costaricensis, is a clinical entity characterized by abdominal pain usually located in the right iliac fossa, accompanied, in some cases, by a tumor-like mass, painful to palpation, which may be confused with a malignancy. Due to the lack of specificity of the symptoms, this parasitosis may also often be confused with an acute abdomen. While in countries like Brazil and Costa Rica the reported incidence reaches up to 28% and 600 cases per year, respectively, the real prevalence and incidence of this parasitosis in the rest of Latin America is unknown, and for some countries it continues to be a rare disease. Since coproparasitological confirmation is not possible for the diagnosis of the infection in humans, an alternative tool has been the development of immunologic techniques for the detection of antibodies in the serum of infected patients. Further, the usefulness of different antigens for an immunological diagnosis has been evaluated using techniques such as ELISA, Western Blot, and immunofluorescence. Among the antigens tested are crude somatic extracts of larval and adult forms, excretion-secretion antigens, low molecular weight fractions, and, most recently, egg antigens. In the field of molecular biology, the development of primers that allow the detection of parasite DNA in the serum of suspected patients have also been reported. This review compiles and analyzes the results of these studies, and emphasizes the need to continue working in the development of a diagnostic test that can elucidate the real impact of abdominal angiostrongyliasis on public health in Latin America.La angiostrongiliasis abdominal, causada por Angiostrongylus costaricensis, es una entidad clínica caracterizada por dolor abdominal localizado usualmente a nivel de la fosa iliaca derecha, acompañado en algunos casos de una masa tumoral, dolorosa a la palpación, que puede llegar a confundirse con un proceso maligno. Debido a la inespecificidad de los síntomas, esta parasitosis también es confundida a menudo con un cuadro de abdomen agudo. Mientras que en países como Brasil y Costa Rica se habla de prevalencias de hasta un 28% y de 600 casos por año, respectivamente, en el resto de Latinoamérica se desconoce la prevalencia e incidencia real de esta parasitosis y para algunos sigue siendo una enfermedad “exótica”. Ante la imposibilidad de un diagnóstico coproparasitológico de la infección en el ser humano, se ha buscado como alternativa el desarrollo de técnicas inmunológicas para la detección de anticuerpos en el suero de personas infectadas. Es así como la utilidad de diferentes antígenos en el diagnóstico inmunológico ha sido evaluada a través de técnicas como ELISA, Western Blot e inmunofluorescencia. Dentro de estos antígenos se incluyen los extractos somáticos crudos de formas larvales y formas adultas, antígenos de excreción-secreción, fracciones de bajo peso molecular y, más recientemente, antígeno de huevecillos. Adicionalmente, ya se ha iniciado la incursión en el campo de la biología molecular con las primeras pruebas que incluyen el diseño de “primers” para la identificación de ADN del parásito en el suero de pacientes sospechosos. Esta revisión compila los resultados de estos estudios y enfatiza la necesidad de seguir trabajando en el desarrollo de una prueba diagnóstica que permita esclarecer el impacto real de la angiostrongiliasis abdominal en el campo de la salud pública en Latinoaméric

Amebas de vida libre como agentes de encefalitis en el ser humano

Este artículo de revisión se enfoca en las amebas de vida libre, un tipo de protozoarios que habitan diferentes tipos de suelo y fuentes de agua. Estos organismos tienen dos estadios evolutivos: trofozoíto y quiste, siendo este último la forma de resistencia que les permite sobrevivir en condiciones adversas del ambiente. La abundancia y diversidad de las amebas de vida libre depende de factores como el clima, pH y disponibilidad de nutrientes. Los trofozoítos se alimentan por fagocitosis y digieren bacterias, hongos y algas ingeridas dentro del fagolisosoma. Además, el artículo señala que algunas de estas amebas tienen la capacidad de comportarse como endoparásitos bajo ciertas condiciones, y que algunas especies de amebas de vida libre pueden ser patógenas para los seres humanos, causando enfermedades como la meningoencefalitis amebiana primaria, la encefalitis granulomatosa amebiana y la queratitis amebiana. Se mencionan algunos casos de infección en humanos y se destaca la importancia de la investigación en este tema para prevenir y tratar estas enfermedades.UCR::Vicerrectoría de Docencia::Salud::Facultad de MicrobiologíaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Primer aislamiento de Balamuthia mandrillaris en Costa Rica

En este estudio se determinó la presencia de Balamuthia mandrillaris en una muestra de polvo procedente de una unidad combinada de emergencia en Costa Rica. Se realizó un muestreo de polvo y agua en 12 unidades combinadas de emergencia instaladas en laboratorios de docencia. Las muestras fueron cultivadas en agar no nutritivo al 1,5% suplementado con Escherichia coli. Las amebas de vida libre aisladas a partir de estas muestras se identificaron morfológicamente y se realizó la caracterización molecular empleando una PCR que amplifica el gen mitocondrial 16S ARNr. La secuenciación del producto de PCR obtenido confirmó el aislamiento de B. mandrillaris, una ameba altamente patógena que produce principalmente cuadros a nivel de sistema nervioso central y piel. A pesar de que el aislamiento de esta especie no se ha ligado a ningún caso clínico en Costa Rica, este reporte es de gran importancia pues representa el primer aislamiento de B. mandrillaris en América Central y el quinto aislamiento a nivel mundial a partir de una muestra ambiental.UCR::Vicerrectoría de Docencia::Salud::Facultad de MicrobiologíaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Primer aislamiento e identificación molecular de Naegleria fowleri en Costa Rica

La meningoencefalitis amebiana primaria, causada por Naegleria fowleri, es un cuadro agudo y fulminante. Esta es una ameba de vida libre (AVL) que presenta amplia distribución a nivel mundial. Presenta una moratildad alta (cerca del 95%) en la mayoría de los casos diagnosticados alrededor del plantea. La Sección de Protozoología Médica de la Faculta de Microbiología fue contactada por el Ministerio de Salud debida a la presencia de una AVL en las aguas termales de la zona de San Carlos, AlajuelaUniversidad de Costa Rica/[803-B4-050]/UCR/Costa RicaUCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

In vitro effects of environmental isolates of Acanthamoeba T4 and T5 over human erythrocytes and platelets

Free-living amoebae of the genus Acanthamoeba have been associated with keratitis and encephalitis. Some factors related to their pathogenic potential have been described, including the release of hydrolytic enzymes, and the adhesion and phagocytosis processes. However, other factors such as their effect over the hemodynamics and microcirculation elements have not been fully investigated. This work determines the in vitro activity of potentially pathogenic environmental isolates of Acanthamoeba genotype T4 and T5 over erythrocytes and platelets. The hemolytic activity (dependent and independent of contact), as well as the production of ADP of ten environmental isolates of Acanthamoeba obtained from dental units, combined emergency showers, dust, and hospital water, were measured. Tests were carried out over erythrocytes in suspension and blood agar plates, incubated at 4 °C, room temperature and 37 °C. Erythrophagocytosis and platelet aggregation assays were also performed. Live trophozoites of all of the isolates tested showed a hemolytic activity that was temperature dependent. Over erythrocytes in suspension, variable hemolysis percentages were obtained: a maximum of 41% and a minimum of 15%. Regarding hemolysis over agar plates, two patterns of hemolysis were observed: double and simple halos. Conditioned medium and crude extracts of trophozoites did not show hemolytic activity. Erythrophagocytosis by Acanthamoeba was also observed; however, no production of ADP was determined by the employed methodology.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Hematología y Trastornos Afines (CIHATA)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Queratitis por Acanthamoeba genotipo T4 en un paciente con queratitis punteada de Thygeson

A case of amoebic keratitis due to Acanthamoeba genotype T4 is reported. The patient is a 74-year old woman with Thygeson’s superficial punctate keratitis. Corneal scrapings were employed for the diagnosis, which included direct microscopic observation, Giemsa stains, cultures in non-nutrient agar and a PCR that amplifies the DF3 fragment of 18S rDNA. The therapeutic use of contact lenses is proposed as the predisposing factor in this case, considering that the patient has a previous damage of the corneal epithelium. This is the first case of amoebic keratitis reported and confirmed in Costa Rica.Universidad de Costa RicaUCR::Vicerrectoría de Docencia::Salud::Facultad de MicrobiologíaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Increasing of Toxoplasma gondii (Coccidia, Sarcocystidae) infections by Trypanosoma lewisi (Kinetoplastida, Trypanosomatidae) in white rats

To demonstrate that T. lewisi infection increases T. gondii multiplication in white rats, groups of five Wistar oc Sprague Dawley rats were inoculated with 106 T. lewisi trypomastigoles and four or seven days later infected with Toxoplasma tachyzoites. Host survival time was monitored, and !he presence of T. gondii was confirmed in all dead rals by studying peritoneal exudate smears and lung tissue sections stained with haematoxylin-eosin. The presence of Toxoplasma cysts or antibodies was ehecked in the brain oí surviving rals. The ¡nerease is observed four days after trypanosome inoculatíon and is dependen! on fat strain, bul not on inoculum size oc cat age. Humoral and cellular factors may have a role in ¡he ¡nerease as has been reported for other experimental infections with African trypanosomes and T. cruzi.To demonstrate that T. lewisi infection increases T. gondii multiplication in white rats, groups of five Wistar oc Sprague Dawley rats were inoculated with 106 T. lewisi trypomastigoles and four or seven days later infected with Toxoplasma tachyzoites. Host survival time was monitored, and !he presence of T. gondii was confirmed in all dead rals by studying peritoneal exudate smears and lung tissue sections stained with haematoxylin-eosin. The presence of Toxoplasma cysts or antibodies was ehecked in the brain oí surviving rals. The ¡nerease is observed four days after trypanosome inoculatíon and is dependen! on fat strain, bul not on inoculum size oc cat age. Humoral and cellular factors may have a role in ¡he ¡nerease as has been reported for other experimental infections with African trypanosomes and T. cruzi