Identificación y caracterización de la proteína del cuello de las roptrias 5 (RON5) en Plasmodium falciparum y determinación de las regiones de unión a glóbulos rojos humanos

87 páginasIn this research, advanced control strategies were designed under the Active Disturbance Rejection Control (ADRC) approach to increase the biomass production in microalgae cultures. For the above, from a control frame of reference, the development was envisaged into two stages, control and optimization. The first stage resulted in three different controllers designs: two ADRC strategies assisted by observer and a Model-Free Control (MFC). In each case, the aim was to guarantee the tracking of the reference signal. In the second stage, the design of two optimization strategies were achieves to increase the biomass production, offline and on-line. Comparing, at a simulation level, these strategies with other existing proposals, the following was found: 1) the ADRC strategies assisted by observer had a few dependence on the model, letting us to work with an approximate model that only required knowing of the system order and the input gain; 2) the off-line optimization, despite maximizing the biomass production, required knowing the model and 3) the proposal that combines MFC with on-line optimization, may act on any microalgae culture since it does not need a model. All the proposals are robust front to disturbances and variation of parameters allowing to increase the biomass production when an optimization strategy is used.La malaria es una de las enfermedades infecciosas más prevalentes y mortales a nivel mundial. Cinco especies de Plasmodium (protozoario intracelular obligado del filo Apicomplexa) infectan al humano, siendo Plasmodium falciparum la especie responsable de las manifestaciones clínicas más severas, con amplia distribución en las zonas tropicales y subtropicales del África Subsahariana. Según los estimados de la Organización Mundial de la Salud (OMS), en el año 2010 se presentaron alrededor de 216 millones de casos de malaria y cerca de un millón de muertes, principalmente de niños menores de 5 años. Esta cifra se ha incrementado en los últimos años, luego de la aparición de variantes del parásito que son resistentes a drogas antimaláricas y por la resistencia a los insectidas por parte del mosquito. Es por lo tanto urgente el desarrollo de medidas de control efectivas que permitan la erradicación de esta parasitosis.Doctorado en BiocienciasDoctor en Biociencia

Parasite-related genetic and epigenetic aspects and host factors influencing plasmodium falciparum invasion of erythrocytes

Malaria, a disease caused by Plasmodium parasites, is widespread throughout tropical and sub-tropical regions worldwide; it mostly affects children and pregnant woman. Eradication has stalled despite effective prevention measures and medication being available for this disease; this has mainly been due to the parasite's resistance to medical treatment and the mosquito vector's resistance to insecticides. Tackling such resistance involves using renewed approaches and techniques for accruing a deep understanding of the parasite's biology, and developing new drugs and vaccines. Studying the parasite's invasion of erythrocytes should shed light on its ability to switch between invasion phenotypes related to the expression of gene sets encoding proteins acting as ligands during target cell invasion, thereby conferring mechanisms for evading a particular host's immune response and adapting to changes in target cell surface receptors. This review considers some factors influencing the expression of such phenotypes, such as Plasmodium's genetic, transcriptional and epigenetic characteristics, and explores some host-related aspects which could affect parasite phenotypes, aiming at integrating knowledge regarding this topic and the possible relationship between the parasite's biology and host factors playing a role in erythrocyte invasion. © 2019 Ararat-Sarria, Patarroyo and Curtidor. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms

PvRON2, a new Plasmodium vivax rhoptry neck antigen

<p>Abstract</p> <p>Background</p> <p>Rhoptries are specialized organelles from parasites belonging to the phylum <it>Apicomplexa</it>; they secrete their protein content during invasion of host target cells and are sorted into discrete subcompartments within rhoptry neck or bulb. This distribution is associated with these proteins' role in tight junction (TJ) and parasitophorous vacuole (PV) formation, respectively.</p> <p>Methods</p> <p><it>Plasmodium falciparum </it>RON2 amino acid sequence was used as bait for screening the codifying gene for the homologous protein in the <it>Plasmodium vivax </it>genome. Gene synteny, as well as identity and similarity values, were determined for <it>ron2 </it>and its flanking genes among <it>P. falciparum</it>, <it>P. vivax </it>and other malarial parasite genomes available at PlasmoDB and Sanger Institute databases. <it>Pvron2 </it>gene transcription was determined by RT-PCR of cDNA obtained from the <it>P. vivax </it>VCG-1 strain. Protein expression and localization were assessed by Western blot and immunofluorescence using polyclonal anti-<it>Pv</it>RON2 antibodies. Co-localization was confirmed using antibodies directed towards specific microneme and rhoptry neck proteins.</p> <p>Results and discussion</p> <p>The first <it>P. vivax </it>rhoptry neck protein (named here <it>Pv</it>RON2) has been identified in this study. <it>Pv</it>RON2 is a 2,204 residue-long protein encoded by a single 6,615 bp exon containing a hydrophobic signal sequence towards the amino-terminus, a transmembrane domain towards the carboxy-terminus and two coiled coil α-helical motifs; these are characteristic features of several previously described vaccine candidates against malaria. This protein also contains two tandem repeats within the interspecies variable sequence possibly involved in evading a host's immune system. <it>Pv</it>RON2 is expressed in late schizonts and localized in rhoptry necks similar to what has been reported for <it>Pf</it>RON2, which suggests its participation during target cell invasion.</p> <p>Conclusions</p> <p>The identification and partial characterization of the first <it>P. vivax </it>rhoptry neck protein are described in the present study. This protein is homologous to <it>Pf</it>RON2 which has previously been shown to be associated with <it>Pf</it>AMA-1, suggesting a similar role for <it>Pv</it>RON2.</p

Parasite-Related Genetic and Epigenetic Aspects and Host Factors Influencing Plasmodium falciparum Invasion of Erythrocytes

Malaria, a disease caused by Plasmodium parasites, is widespread throughout tropical and sub-tropical regions worldwide; it mostly affects children and pregnant woman. Eradication has stalled despite effective prevention measures and medication being available for this disease; this has mainly been due to the parasite's resistance to medical treatment and the mosquito vector's resistance to insecticides. Tackling such resistance involves using renewed approaches and techniques for accruing a deep understanding of the parasite's biology, and developing new drugs and vaccines. Studying the parasite's invasion of erythrocytes should shed light on its ability to switch between invasion phenotypes related to the expression of gene sets encoding proteins acting as ligands during target cell invasion, thereby conferring mechanisms for evading a particular host's immune response and adapting to changes in target cell surface receptors. This review considers some factors influencing the expression of such phenotypes, such as Plasmodium's genetic, transcriptional and epigenetic characteristics, and explores some host-related aspects which could affect parasite phenotypes, aiming at integrating knowledge regarding this topic and the possible relationship between the parasite's biology and host factors playing a role in erythrocyte invasion

Conserved binding regions provide the clue for peptide-based vaccine development: A chemical perspective

Synthetic peptides have become invaluable biomedical research and medicinal chemistry tools for studying functional roles, i.e., binding or proteolytic activity, naturally-occurring regions' immunogenicity in proteins and developing therapeutic agents and vaccines. Synthetic peptides can mimic protein sites; their structure and function can be easily modulated by specific amino acid replacement. They have major advantages, i.e., they are cheap, easily-produced and chemically stable, lack infectious and secondary adverse reactions and can induce immune responses via T- and B-cell epitopes. Our group has previously shown that using synthetic peptides and adopting a functional approach has led to identifying Plasmodium falciparum conserved regions binding to host cells. Conserved high activity binding peptides' (cHABPs) physicochemical, structural and immunological characteristics have been taken into account for properly modifying and converting them into highly immunogenic, protection-inducing peptides (mHABPs) in the experimental Aotus monkey model. This article describes stereo-electron and topochemical characteristics regarding major histocompatibility complex (MHC)-mHABP-T-cell receptor (TCR) complex formation. Some mHABPs in this complex inducing long-lasting, protective immunity have been named immune protection-inducing protein structures (IMPIPS), forming the subunit components in chemically synthesized vaccines. This manuscript summarizes this particular field and adds our recent findings concerning intramolecular interactions (H-bonds or-interactions) enabling proper IMPIPS structure as well as the peripheral flanking residues (PFR) to stabilize the MHCII-IMPIPS-TCR interaction, aimed at inducing long-lasting, protective immunological memory. © 2017 by the authors. Licensee MDPI, Basel, Switzerland.1

Using the PfEMP1 Head Structure Binding Motif to Deal a Blow at Severe Malaria

Plasmodium falciparum (Pf) malaria causes 200 million cases worldwide, 8 million being severe and complicated leading to similar to 1 million deaths and similar to 100,000 abortions annually. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) has been implicated in cytoadherence and infected erythrocyte rosette formation, associated with cerebral malaria; chondroitin sulphate-A attachment and infected erythrocyte sequestration related to pregnancy-associated malaria and other severe forms of disease. An endothelial cell high activity binding peptide is described in several of this similar to 300 kDa hypervariable protein's domains displaying a conserved motif (GACxPxRRxxLC); it established H-bonds with other binding peptides to mediate red blood cell group A and chondroitin sulphate attachment. This motif (when properly modified) induced PfEMP1-specific strain-transcending, fully-protective immunity for the first time in experimental challenge in Aotus monkeys, opening the way forward for a long sought-after vaccine against severe malaria

Mycobacterium tuberculosis Rv0679c protein sequences involved in host-cell infection: Potential TB vaccine candidate antigen

Hasta la fecha, la función de muchas proteínas de membrana hipotéticas de Mycobacterium tuberculosis aún se desconoce y su participación en las interacciones patógeno-huésped aún no se ha definido claramente. En este estudio, se evaluó la actividad biológica de los péptidos derivados de la proteína de membrana hipotética Rv0679c de M. tuberculosis y su participación en las interacciones patógeno-huésped. La transcripción del gen Rv0679c se estudió en 26 Mycobacteriumspp. Son. Los anticuerpos generados contra los supuestos epítopos de células B de Rv0679c se usaron en ensayos de inmunotransferencia y microscopía inmunoelectrónica. Los péptidos sintéticos que abarcan toda la longitud de la proteína fueron probados por su capacidad para unirse a las células A549 y U937. Los péptidos de unión de alta actividad (HABP) identificados en Rv0679c se probaron para determinar su capacidad para inhibir la invasión de micobacterias en las células.To date, the function of many hypothetical membrane proteins of Mycobacterium tuberculosis is still unknown and their involvement in pathogen-host interactions has not been yet clearly defined. In this study, the biological activity of peptides derived from the hypothetical membrane protein Rv0679c of M. tuberculosis and their involvement in pathogen-host interactions was assessed. Transcription of the Rv0679c gene was studied in 26 Mycobacterium spp. Strains. Antibodies raised against putative B-cell epitopes of Rv0679c were used in Western blot and immunoelectron microscopy assays. Synthetic peptides spanning the entire length of the protein were tested for their ability to bind to A549 and U937 cells. High-activity binding peptides (HABPs) identified in Rv0679c were tested for their ability to inhibit mycobacterial invasion into cells

Sexual forms obtained in a continuous in vitro cultured Colombian strain of Plasmodium falciparum (FCB2)

Background: The epidemiological control of malaria has been hampered by the appearance of parasite resistance to anti-malarial drugs and by the resistance of mosquito vectors to control measures. This has also been associated with weak transmission control, mostly due to poor control of asymptomatic patients associated with host-vector transmission. This highlights the importance of studying the parasite's sexual forms (gametocytes) which are involved in this phase of the parasite's life-cycle. Some African and Asian strains of Plasmodium falciparum have been fully characterized regarding sexual forms' production; however, few Latin-American strains have been so characterized. This study was aimed at characterizing the Colombian FCB2 strain as a gametocyte producer able to infect mosquitoes. Methods: Gametocyte production was induced in in vitro cultured P. falciparum FCB2 and 3D7 strains. Pfap2g and Pfs25 gene expression was detected in FCB2 strain gametocyte culture by RT-PCR. Comparative analysis of gametocytes obtained from both strains was made (counts and morphological changes). In vitro zygote formation from FCB2 gametocytes was induced by incubating a gametocyte culture sample at 27 °C for 20 min. A controlled Anopheles albimanus infection was made using an artificial feed system with cultured FCB2 gametocytes (14-15 days old). Mosquito midgut dissection was then carried out for analyzing oocysts. Results: The FCB2 strain expressed Pfap2g, Pfs16, Pfg27/25 and Pfs25 sexual differentiation-related genes after in vitro sexual differentiation induction, producing gametocytes that conserved the expected morphological features. The amount of FCB2 gametocytes produced was similar to that from the 3D7 strain. FCB2 gametocytes were differentiated into zygotes and ookinetes after an in vitro low-temperature stimulus and infected An. albimanus mosquitoes, developing to oocyst stage. Conclusions: Even with the history of long-term FCB2 strain in vitro culture maintenance, it has retained its sexual differentiation ability. The gametocytes produced here preserved these parasite forms' usual characteristics and An. albimanus infection capability, thus enabling its use as a tool for studying sexual form biology, An. albimanus infection comparative analysis and anti-malarial drug and vaccine development. © 2020 The Author(s)

PvRON2, a new Plasmodium vivax rhoptry neck antigen

Background: Rhoptries are specialized organelles from parasites belonging to the phylum Apicomplexa; they secrete their protein content during invasion of host target cells and are sorted into discrete subcompartments within rhoptry neck or bulb. This distribution is associated with these proteins’ role in tight junction (TJ) and parasitophorous vacuole (PV) formation, respectively. Methods: Plasmodium falciparum RON2 amino acid sequence was used as bait for screening the codifying gene for the homologous protein in the Plasmodium vivax genome. Gene synteny, as well as identity and similarity values, were determined for ron2 and its flanking genes among P. falciparum, P. vivax and other malarial parasite genomes available at PlasmoDB and Sanger Institute databases. Pvron2 gene transcription was determined by RT-PCR of cDNA obtained from the P. vivax VCG-1 strain. Protein expression and localization were assessed by Western blot and immunofluorescence using polyclonal anti-PvRON2 antibodies. Co-localization was confirmed using antibodies directed towards specific microneme and rhoptry neck proteins. Results and discussion: The first P. vivax rhoptry neck protein (named here PvRON2) has been identified in this study. PvRON2 is a 2,204 residue-long protein encoded by a single 6,615 bp exon containing a hydrophobic signal sequence towards the amino-terminus, a transmembrane domain towards the carboxy-terminus and two coiled coil a-helical motifs; these are characteristic features of several previously described vaccine candidates against malaria. This protein also contains two tandem repeats within the interspecies variable sequence possibly involved in evading a host’s immune system. PvRON2 is expressed in late schizonts and localized in rhoptry necks similar to what has been reported for PfRON2, which suggests its participation during target cell invasion. Conclusions: The identification and partial characterization of the first P. vivax rhoptry neck protein are described in the present study. This protein is homologous to PfRON2 which has previously been shown to be associated with PfAMA-1, suggesting a similar role for PvRON2

Mycobacterium tuberculosis Rv0679c protein sequences involved in host-cell infection: Potential TB vaccine candidate antigen

BACKGROUND: To date, the function of many hypothetical membrane proteins of Mycobacterium tuberculosis is still unknown and their involvement in pathogen-host interactions has not been yet clearly defined. In this study, the biological activity of peptides derived from the hypothetical membrane protein Rv0679c of M. tuberculosis and their involvement in pathogen-host interactions was assessed. Transcription of the Rv0679c gene was studied in 26 Mycobacterium spp. Strains. Antibodies raised against putative B-cell epitopes of Rv0679c were used in Western blot and immunoelectron microscopy assays. Synthetic peptides spanning the entire length of the protein were tested for their ability to bind to A549 and U937 cells. High-activity binding peptides (HABPs) identified in Rv0679c were tested for their ability to inhibit mycobacterial invasion into cells. RESULTS: The gene encoding Rv0679c was detected in all strains of the M. tuberculosis complex (MTC), but was only transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra and M. africanum. Anti-Rv0679c antibodies specifically recognized the protein in M. tuberculosis H37Rv sonicate and showed its localization on mycobacterial surface. Four HABPs inhibited invasion of M. tuberculosis to target cells by up to 75%. CONCLUSIONS: The results indicate that Rv0679c HABPs and in particular HABP 30979 could be playing an important role during M. tuberculosis invasion of host cells, and therefore could be interesting research targets for studies aimed at developing strategies to control tuberculosis