Cysteine proteinase C1A paralog profiles correspond with phylogenetic lineages of pathogenic piroplasmids

Piroplasmid parasites comprising of Babesia, Theileria, and Cytauxzoon are transmitted by ticks to farm and pet animals and have a significant impact on livestock industries and animal health in tropical and subtropical regions worldwide. In addition, diverse Babesia spp. infect humans as opportunistic hosts. Molecular phylogeny has demonstrated at least six piroplasmid lineages exemplified by B. microti, B. duncani, C. felis, T. equi, Theileria sensu stricto (T. annulata, T. parva, and T. orientalis) and Babesia sensu stricto (B. bovis, B. bigemina, and B. ovis). C1A cysteine-proteinases (C1A-Cp) are papain-like enzymes implicated in pathogenic and vital steps of the parasite life cycle such as nutrition and host cell egress. An expansion of C1A-Cp of T. annulata and T. parva with respect to B. bovis and B. ovis was previously described. In the present work, C1A-Cp paralogs were identified in available genomes of species pertaining to each piroplasmid lineage. Phylogenetic analysis revealed eight C1A-Cp groups. The profile of C1A-Cp paralogs across these groups corroborates and defines the existence of six piroplasmid lineages. C. felis, T. equi and Theileria s.s. each showed characteristic expansions into extensive families of C1A-Cp paralogs in two of the eight groups. Underlying gene duplications have occurred as independent unique evolutionary events that allow distinguishing these three piroplasmid lineages. We hypothesize that C1A-Cp paralog families may be associated with the advent of the schizont stage. Differences in the invertebrate tick host specificity and/or mode of transmission in piroplasmid lineages might also be associated with the observed C1A-Cp paralog profiles

Novedoso desarrollo biotecnológico : alimentos con menos colesterol

Fil: Nudel, Clara. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Catedra de Microbiología Industrial y Biotecnología; Argentina.Fil: Nusblat, Alejandro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Cátedra de Microbiología Industrial y Biotecnología; Argentina.Fil: Valcarce, German. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Florin- Christensen, Jorge. Universidad de Buenos Aires. Facultad de Medicina; Argentina.Investigadores del Conicet y de la Facultad de Farmacia y Bioquímica de la UBA desarrollaron un\nmicroorganismo unicelular (Tetrahymena) que al ser incorporado a alimentos como la leche y el huevo\nles reduce el colesterol en un 90% transformándolo en provitamina D

Release of lysosomal enzymes in Tetrahymena: a Ca²⁺-dependent secretory process

The ciliate Tetrahymena thermophila releases lysosomal enzymes into nutrient and starvation media. We show here that this process occurs selectively, i.e. without leakage of cytoplasmic components, as indicated by lack of release of isocitrate dehydrogenase, a cytosolic enzyme with high activity in Tetrahymena . The role of intracellular Ca²⁺ in the process was also investigated. The Ca²⁺ ionophore A23187 has strong stimulatory effects on this release. Ionophore stimulation is maximal in the presence of extracellular Ca²⁺ but can occur also in its absence. Quin 2 fluorescence measurements indicate that intracellular Ca²⁺ increases in both cases. Mg²⁺ completely prevents the stimulatory effects of A23187. Ionomycin, another Ca²⁺ ionophore, also stimulates lysosomal enzyme release with a maximal response in the presence of extracellular Ca²⁺ . Measurements of extracellular isocitrate dehydrogenase showed that ionophore-stimulated lysosomal enzyme release can take place without leakage of cytoplasmic components. The observations that divalent cation ionophores stimulate selective lysosomal enzyme release and that this effect is strongest in the presence of external Ca²⁺ indicate that this cation plays a crucial role in the control of this process in Tetrahymena . Together with other observations they support the view that a subpopulation of Tetrahymena lysosomes has properties like those of secretory vesicles.Centro de Estudios Parasitológicos y de Vectore

Development of fluorogenic probe-based PCR assays for the detection and quantification of bovine piroplasmids.

This paper reports two new quantitative PCR (qPCR) assays, developed in an attempt to improve the detection of bovine piroplasmids. The first of these techniques is a duplex TaqMan assay for the simultaneous diagnosis of Babesia bovis and B. bigemina. This technique is ideal for use in South America where bovids harbour no theilerids. The second technique, which is suitable for the diagnosis of both babesiosis and theileriosis worldwide, involves fluorescence resonance energy transfer (FRET) probes. In FRET assays, Babesia bovis, B. divergens, Babesia sp. (B. major or B. bigemina), Theileria annae and Theileria sp. were all identifiable based on the melting temperatures of their amplified fragments. Both techniques provided linear calibration curves over the 0.1fg/microl to 0.01ng/microl DNA range. The assays showed good sensitivity and specificity. To assess their performance, both procedures were compared in two separate studies: the first was intended to monitor the experimental infection of calves with B. bovis and the second was a survey where 200 bovid/equine DNA samples from different countries were screened for piroplasmids. Comparative studies showed that duplex TaqMan qPCR was more sensitive than FRET qPCR in the detection of babesids

Rhipicephalus (Boophilus) Microplus Ticks can Complete their Life Cycle on the Water Buffalo (Bubalus bubalis)

Rhipicephalus (Boophilus) microplus is considered one of the most important ectoparasites of cattle worldwide. Due to the increase in the number of water buffaloes (Bubalus bubalis) in R. microplus-infested areas, this study was designed to determine whether these ruminants are able to sustain the complete tick life cycle. To this aim, a seven-month old water buffalo of the Mediterranean breed and a Holstein bovine of the same age, both tick-naïve, were infested with R. microplus tick larvae, and the parasitic and non-parasitic tick stages were analyzed and compared. The studied parameters include the number of recovered engorged females, the time points at which the first and last engorged females fell to the ground; the pre-oviposition duration, the percentage of hatching and the reproductive efficiency index. No statistically significant differences were found between the buffalo and the bovine in all parameters measured. It was concluded that the water buffalo can act as a suitable reservoir for R. microplus ticks. These results should be taken into account when implementing tick control and eradication campaigns in water buffalo grazing lands

Release of lysosomal enzymes in Tetrahymena: a Ca²⁺-dependent secretory process

The ciliate Tetrahymena thermophila releases lysosomal enzymes into nutrient and starvation media. We show here that this process occurs selectively, i.e. without leakage of cytoplasmic components, as indicated by lack of release of isocitrate dehydrogenase, a cytosolic enzyme with high activity in Tetrahymena . The role of intracellular Ca²⁺ in the process was also investigated. The Ca²⁺ ionophore A23187 has strong stimulatory effects on this release. Ionophore stimulation is maximal in the presence of extracellular Ca²⁺ but can occur also in its absence. Quin 2 fluorescence measurements indicate that intracellular Ca²⁺ increases in both cases. Mg²⁺ completely prevents the stimulatory effects of A23187. Ionomycin, another Ca²⁺ ionophore, also stimulates lysosomal enzyme release with a maximal response in the presence of extracellular Ca²⁺ . Measurements of extracellular isocitrate dehydrogenase showed that ionophore-stimulated lysosomal enzyme release can take place without leakage of cytoplasmic components. The observations that divalent cation ionophores stimulate selective lysosomal enzyme release and that this effect is strongest in the presence of external Ca²⁺ indicate that this cation plays a crucial role in the control of this process in Tetrahymena . Together with other observations they support the view that a subpopulation of Tetrahymena lysosomes has properties like those of secretory vesicles.Centro de Estudios Parasitológicos y de Vectore

Caracterización de un sistema de enzimas lipolíticas del medio de cultivo de Tetrahymena

En esta Tesis se describe el hallazgo de actividad hemolítica, así comotambién de tres actividades lipolíticas en el medio extracelular delprotozoo ciliado Ietrahymena thermophila. Las enzimas halladas son unafosfolipasa C (EC.3.1.4.3), una fosfolipasa A, (EC.3.1.1.32) y una triacilglicerollipasa (EC.3.1.1.3). Estas enzimas fueron purificadasmediante precipitación fraccionada con sulfato de amonio y cromatografíade intercambio iónico, de filtración en geles y de interacción hidrofóbicaen decil Sepharose 4B. Se encontró así que la fosfolipasa Ccopurífica con actividad hemolítica, no así la triacilglicerol lipasa nila fosfolipasa A, aunque esta última es capaz de acelerar la acciónhemolítica de la fosfolipasa C. Se halló además un segundo factorhemolítico no asociado a fosfolipasas, cuya acción es inhibida porcolesterol y que, junto con la fosfolipasa C, puede dar cuenta de laacción hemolítica causada por el medio extracelular de Tetrahymena. Estesegundo factor podría actuar en forma semejante a ciertas citolisinasbacterianas que causan hemólisis por interacción con el colesterol delas membranas. Las tres enzimas lipolíticas fueron caracterizadas y suspropiedades indican que provienen del sistema lisosomal. El hallazgo delas actividades aquí descriptas proporciona una explicación para elenigmático rol de los fosfonolípidos, un tipo especial de fosfolípidosque predominan en la membrana plasmática de Tetrahymen. Estos compuestospresentan un enlace directo carbono-fósforo que los hace particularmenteresistentes a la hidrólisis enzimática por fosfolipasa C. Poseen,además, un enlace éter en la posición sn-1, que les confiere resistenciaa la acción de fosfolipasa A. Es decir, Tetrahymena, una célula carentede pared celular u otras estructuras protectoras, sería capaz de tolerarsus propias secreciones líticas gracias a este tipo peculiar de fosfolípidos. Otras observaciones presentadas aquí pueden estar relacionadascon particularidades de las membranasde este ciliado. Así debe destacarsela baja proporción de triacilglicerol en la membrana plasmática,contrastando con lo observado para membranas internas de Tetrahymena. Este hecho puede estar vinculado con la secreción de la triacilglicerollipasa. Otro aspecto notable es que esta célula carece totalmente decolesterol, el que es reemplazado por tetrahymanol. Esto puede explicarque el segundo factor hemolitico hallado no actúe sobre la membrana de Tetrahymena y sí sobre la de los eritrocitos. En esta Tesis se proponeque la secreción de enzimas y las particularidades de la membranarepresentan una coadaptación que puede conferir a estos ciliados unaventaja evolutiva, al ser capaces de lisar otras células siendo ellosmismos resistentes. Otros experimentos presentados aquí sugieren, porúltimo, que las actividades lipolíticas secretadas pueden desempeñar unrol destacado en la digestión extracelular e incorporación de fosfolípidosexógenos en este ciliado.Fil: Florin-Christensen, Jorge. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Ocellatin-PT antimicrobial peptides: high-resolution microscopy studies in antileishmania models and interactions with mimetic membrane systems

Although the mechanism of action of antimicrobial peptides (AMPs) is not clear, they can interact electrostatically with the cell membranes of microorganisms. New ocellatin-PT peptides were recently isolated from the skin secretion of Leptodactylus pustulatus. The secondary structure of these AMPs and their effect on Leishmania infantum cells, and on different lipid surface models was characterized in this work. The results showed that all ocellatin-PT peptides have an -helix structure and five of them (PT3, PT4, PT6 to PT8) have leishmanicidal activity; PT1 and PT2 affected the cellular morphology of the parasites and showed greater affinity for leishmania and bacteria-mimicking lipid membranes than for those of mammals. The results show selectivity of ocellatin-PTs to the membranes of microorganisms and the applicability of biophysical methods to clarify the interaction of AMPs with cell membranes.This work was partially supported by grants from INCT Nanobiotecnologia and PVE Project (MCT/CNPq), the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET), and the Agencia Nacional de Promocion Científica y Tecnologica (ANPCyT). M.M.M.is a researcher at CONICET. This work has also been supported through project UID/MULTI/04378/2013-POCI/01/0145/FEDER/007728 with financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020. Scanning electron microscopy was carried out at Centro de Materiais da Universidade do Porto, CEMUP. Peter Eaton is supported by a Ciência sem Fronteiras grant via CNPq, and his lab work is supported by financial support from FCT/MEC through national funds and co-financed by FEDER, under the partnership agreement PT2020. Alexandra Pl acido and Ana Georgina Gomes-Alves are grateful to FCT for their grants SFRH/BD/97995/2013 and SFRH/BD/93766/2013, financed by POPH–QREN–Tipologia 4.1–Formação Avançada, subsidized by Fundo Social Europeu and Ministério da Ciência, Tecnologia e Ensino Superior. Nuno Vale thanks Programa Operacional Regional do Norte (ON.2) and Faculdade de Ciências da Universidade do Porto (FCUP) for co-funding refurbishment of the Porto Peptide Synthesis Facility (POPUP) through operation NORTE-07-0162-FEDER000111. NV thanks Fundação para a Ciência e Tecnologia (FCT, Portugal) and FEDER (European Union) for funding through project grant IF/00092/2014. Work in AMT laboratory was supported by Project “NORTE-07-0124-FEDER-000002-Host-Pathogen Interactions” cofunded by Programa Operacional Regional do Norte under QREN, through FEDER and FCT. None of the funding bodies were involved in study design, in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication

In Silico Survey and Characterization of Babesia microti Functional and Non-Functional Proteases

Human babesiosis caused by the intraerythrocytic apicomplexan Babesia microti is an expanding tick-borne zoonotic disease that may cause severe symptoms and death in elderly or immunocompromised individuals. In light of an increasing resistance of B. microti to drugs, there is a lack of therapeutic alternatives. Species-specific proteases are essential for parasite survival and possible chemotherapeutic targets. However, the repertoire of proteases in B. microti remains poorly investigated. Herein, we employed several combined bioinformatics tools and strategies to organize and identify genes encoding for the full repertoire of proteases in the B. microti genome. We identified 64 active proteases and 25 nonactive protease homologs. These proteases can be classified into cysteine (n = 28), serine (n = 21), threonine (n = 14), asparagine (n = 7), and metallopeptidases (n = 19), which, in turn, are assigned to a total of 38 peptidase families. Comparative studies between the repertoire of B. bovis and B. microti proteases revealed differences among sensu stricto and sensu lato Babesia parasites that reflect their distinct evolutionary history. Overall, this data may help direct future research towards our understanding of the biology and pathogenicity of Babesia parasites and to explore proteases as targets for developing novel therapeutic interventions.Instituto de PatobiologíaFil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Florin-Christensen, Monica. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wieser, Sarah Nathaly. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina.Fil: Wieser, Sarah Nathaly. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Suarez, Carlos E. USDA-ARS. Animal Disease Research Unit; Estados UnidosFil: Suarez, Carlos E. Washington State University. Department of Veterinary Microbiology and Pathology; Estados UnidosFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin