Extracellular vesicles release from protozoa parasite and animal model.

Diseases caused by protozoan parasites, such as leishmaniasis, trypanosomiasis, and malaria, are highly complex and together continue to cause high annual morbidity and mortality. The search for new compounds in environmental biodiversity, repositioning known drugs, and developing vaccines using old and innovative technologies have been employed to discover vaccines and new and alternative treatments. Extracellular vesicles (EVs) can carry parasite antigens, creating a new possibility to develop an effective and affordable platform for treatment, vaccines, and drug delivery. Thus, the evaluation of EVs in animal models can and should be explored among the countless biomedical applications. Herein, we will address the concept of EVs, their acquisition and characterization in protozoan parasite models, and the primary studies using these vesicles in therapeutic applications.

Insights into Peptidyl-Prolyl cis-trans Isomerases from Clinically Important Protozoans: From Structure to Potential Biotechnological Applications.

Peptidyl-prolyl cis/trans isomerases (PPIases) are present in a wide variety of microorganisms, including protozoan parasites such as Trypanosoma cruzi, Trypanosoma brucei, Trichomonas vaginalis, Leishmania major, Leishmania donovani, Plasmodium falciparum, Plasmodium vivax, Entamoeba histolytica, Giardia intestinalis, Cryptosporidium parvum, and Cryptosporidium hominis, all of which cause important neglected diseases. PPIases are classified as cyclophilins, FKBPs, or parvulins and play crucial roles in catalyzing the cis-trans isomerization of the peptide bond preceding a proline residue. This activity assists in correct protein folding. However, experimentally, the biological structure-function characterization of PPIases from these protozoan parasites has been poorly addressed. The recombinant production of these enzymes is highly relevant for this ongoing research. Thus, this review explores the structural diversity, functions, recombinant production, activity, and inhibition of protozoan PPIases. We also highlight their potential as biotechnological tools for the in vitro refolding of other recombinant proteins from these parasites. These applications are invaluable for the development of diagnostic and therapeutic tools.

Entamoeba histolytica: EhADH, an Alix Protein, Participates in Several Virulence Events through Its Different Domains.

Entamoeba histolytica is the protozoan causative of human amoebiasis. The EhADH adhesin (687 aa) is a protein involved in tissue invasion, phagocytosis and host-cell lysis. EhADH adheres to the prey and follows its arrival to the multivesicular bodies. It is an accessory protein of the endosomal sorting complexes required for transport (ESCRT) machinery. Here, to study the role of different parts of EhADH during virulence events, we produced trophozoites overexpressing the three domains of EhADH, Bro1 (1-400 aa), Linker (246-446 aa) and Adh (444-687 aa) to evaluate their role in virulence. The TrophozBro11-400 slightly increased adherence and phagocytosis, but these trophozoites showed a higher ability to destroy cell monolayers, augment the permeability of cultured epithelial cells and mouse colon, and produce more damage to hamster livers. The TrophozLinker226-446 also increased the virulence properties, but with lower effect than the TrophozBro11-400. In addition, this fragment participates in cholesterol transport and GTPase binding. Interestingly, the TrophozAdh444-687 produced the highest effect on adherence and phagocytosis, but it poorly influenced the monolayers destruction; nevertheless, they augmented the colon and liver damage. To identify the protein partners of each domain, we used recombinant peptides. Pull-down assays and mass spectrometry showed that Bro1 domain interplays with EhADH, Gal/GalNAc lectin, EhCPs, ESCRT machinery components and cytoskeleton proteins. While EhADH, ubiquitin, EhRabB, EhNPC1 and EhHSP70 were associated to the Linker domain, and EhADH, EhHSP70, EhPrx and metabolic enzymes interacted to the Adh domain. The diverse protein association confirms that EhADH is a versatile molecule with multiple functions probably given by its capacity to form distinct molecular complexes.

Nanopore Long Read DNA Sequencing of Protozoan Parasites: Hybrid Genome Assembly of Trypanosoma cruzi.

Due to highly repetitive genome sequences, short-read-based Trypanosoma cruzi genomes are extremely fragmented. Contiguous trypanosomatid genomes assemblies have resulted in the advent of third-generation sequencing technologies. Long reads span several to hundreds of kbps allowing to correct assemblies of repeated and low complexity DNA regions. However, these techniques present higher error rates. Hybrid assembly strategies that combine error-prone long reads with much more accurate Illumina short reads represent a very convenient approach for enhancing genome completeness. Here, we describe how to perform a hybrid assembly for genomic analysis of protozoan pathogens using Illumina and Oxford Nanopore sequencing.

Leishmania: manipulation of signaling pathways to inhibit host cell apoptosis.

The maintenance of homeostasis in living systems requires the elimination of unwanted cells which is performed, among other mechanisms, by type I cell death or apoptosis. This type of programmed cell death involves several morphological changes such as cytoplasm shrinkage, chromatin condensation (pyknosis), nuclear fragmentation (karyorrhexis), and plasma membrane blebbing that culminate with the formation of apoptotic bodies. In addition to the maintenance of homeostasis, apoptosis also represents an important defense mechanism for cells against intracellular microorganisms. In counterpart, diverse intracellular pathogens have developed a wide array of strategies to evade apoptosis and persist inside cells. These strategies include the manipulation of signaling pathways involved in the inhibition of apoptosis where mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) play a key role. Leishmania is an intracellular protozoan parasite that causes a wide spectrum of diseases known as leishmaniasis. This parasite displays different strategies, including apoptosis inhibition, to down-regulate host cell defense mechanisms in order to perpetuate infection.

Antagonistic effects of native strains of the soil fungus Paecilomyces against gastrointestinal nematode and protozoan parasites of pigs in Panama.

A variety of gastrointestinal parasites naturally infect domestic pigs in Panama which may also occur as zoonotic infections in humans. Anthelmintic drug treatment, including mass drug administration, can lead to drug resistance, reflecting a need for alternatives. The objectives of this exploratory and observational study were: (1) to isolate and cultivate natives species of Paecilomyces from natural soils in Panama, and (2) to evaluate isolated strains for their capacity to parasitize endemic gastrointestinal nematode and protozoan parasites recovered from naturally infected domestic pigs by observing cultures for spore adhesion and hyphae penetration phases. Using microcultivation and inoculation techniques, four strains of Paecilomyces were isolated from three locations in Panama, out of which three successfully adhered to and penetrated free-living stages (eggs, cysts and oocysts) of Balantidium suis, coccidia, Trichuris suis and hookworm. To our knowledge, this is the first published report of a nematophagous fungus such as Paecilomyces successfully infecting this range of gastrointestinal parasites, particularly protozoan parasites.

First record of dark leathery surface of geoduck clams in Panopea globosa.

Dark Leathery Surface of Geoduck Clams (LSGC) is an alteration that affects the periostracum of the mantle and siphon of Panopea generosa from the northwest coast of Canada and Mexico. This alteration affects commercialization and possibly the survival of the clams. The cause of LSGC is unknown but has been correlated with presence of fungi and protozoans. We detected a similar alteration in Panopea globosa from Baja California, Mexico and the histophagous ciliate Uronema marinum was isolated from affected siphon tissue. U. marinum was identified by its morphology and by genetic analysis of the gene 18S rRNA. This is the first record of LSGC in P. globosa and the first identification of a histophagous protozoan associated with it.

Intracellular Ca2+ Signaling in Protozoan Parasites: An Overview with a Focus on Mitochondria.

Ca2+ signaling has been involved in controling critical cellular functions such as activation of proteases, cell death, and cell cycle control. The endoplasmatic reticulum plays a significant role in Ca2+ storage inside the cell, but mitochondria have long been recognized as a fundamental Ca2+ pool. Protozoan parasites such as Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma cruzi display a Ca2+ signaling toolkit with similarities to higher eukaryotes, including the participation of mitochondria in Ca2+-dependent signaling events. This review summarizes the most recent knowledge in mitochondrial Ca2+ signaling in protozoan parasites, focusing on the mechanism involved in mitochondrial Ca2+ uptake by pathogenic protists.

Synthetic ligustrazine based cyclohexanone and oxime analogs as Anti-Trypanosoma and Anti-Leishmanial agentes

In the present study a series of 34 synthetic ligustrazine-containing α, ß-Unsaturated carbonyl-based compounds and oximes, recognized as anticancer compounds were assessed against protozoa of the Trypanosoma and Leishmania species. Ligustrazine, chemically known as tetramethylpyrazine (TMP), was selected as the core moiety for the synthesis of α, ß-Unsaturated carbonyl-based compounds and these compounds were selected as precursors for the synthesis of new oximes. Some derivates, including 5f and 6i, showed multiple activities against all tested strains. In particular compounds 5f and 8o are the most potent and they are, therefore, potential candidates for trypanosomiasis and leishmaniasis

Extracellular vesicles in infectious diseases caused by protozoan parasites in buffaloes.

BACKGROUND: Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. METHODS: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. RESULTS: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. CONCLUSIONS: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.

Extracellular vesicles in infectious diseases caused by protozoan parasites in buffaloes

Background: Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. Methods: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. Results: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. Conclusions: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.(AU)

Extracellular vesicles in infectious diseases caused by protozoan parasites in buffaloes

Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. Methods: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. Results: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. Conclusions: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.(AU)

Nanopore Sequencing Significantly Improves Genome Assembly of the Protozoan Parasite Trypanosoma cruzi.

Chagas disease was described by Carlos Chagas, who first identified the parasite Trypanosoma cruzi from a 2-year-old girl called Berenice. Many T. cruzi sequencing projects based on short reads have demonstrated that genome assembly and downstream comparative analyses are extremely challenging in this species, given that half of its genome is composed of repetitive sequences. Here, we report de novo assemblies, annotation, and comparative analyses of the Berenice strain using a combination of Illumina short reads and MinION long reads. Our work demonstrates that Nanopore sequencing improves T. cruzi assembly contiguity and increases the assembly size in ∼16 Mb. Specifically, we found that assembly improvement also refines the completeness of coding regions for both single-copy genes and repetitive transposable elements. Beyond its historical and epidemiological importance, Berenice constitutes a fundamental resource because it now constitutes a high-quality assembly available for TcII (clade C), a prevalent lineage causing human infections in South America. The availability of Berenice genome expands the known genetic diversity of these parasites and reinforces the idea that T. cruzi is intraspecifically divided in three main clades. Finally, this work represents the introduction of Nanopore technology to resolve complex protozoan genomes, supporting its subsequent application for improving trypanosomatid and other highly repetitive genomes.

Molecular detection and genotyping of pathogenic protozoan parasites in raw and treated water samples from southwest Colombia.

BACKGROUND: Protozoan parasites such as Giardia duodenalis, Cryptosporidium spp., Cyclospora cayetanensis, Toxoplasma gondii and Entamoeba histolytica represent a great challenge to the systems producing water for human consumption because their cystic forms are persistent in the environment and resist to the disinfection methods conventionally used for their control. In this study, we investigated the presence of these protozoan pathogens in both raw and treated water samples used for the production of drinking water in Nariño Department, southwest Colombia. We collected 110 water samples (10 lof each sample) and analyzed them with real-time PCR (qPCR). qPCR-positive samples were genotyped with PCR and DNA sequencing. RESULTS: Giardia duodenalis was detected in 35/110 (31.8%) of the samples and Cryptosporidium spp. in 9/110 (8.2%) of the samples; no sample was positive for T. gondii, E. histolytica or C. cayetanensis. Giardia duodenalis was detected in samples of both raw water (Drinking Water Treatment Plants (DWTP): 47.83%;Drinking Water Rural Plants (DWRP): 18.42%) and water collected either after conventional physicochemical treatment (26.09%) or after disinfection by chlorine (50%), whereas Cryptosporidium spp. were only detected in raw waters (DWTP: 17.39%; DWRP: 13.16%). The two pathogens were detected in both types of treatment plants supplying water to urban areas and to rural zones. Analysis of gdh and tpi markers identified assemblages AI, AII and H of G. duodenalis, while analysis of the small subunit rRNA and gp60 markers of Cryptosporidium-positive samples identified C. parvum (Subtype IIcA5G3c), C. galli, C. molnari, Cryptosporidium sp. genotype II of bats and Cryptosporidium sp. genotype VIII of birds. CONCLUSIONS: The results obtained demonstrate the presence of protozoan parasites in the water of the study region, and the need to improve the surveillance systems for these pathogens and identify the corresponding sources of contamination.

Probing the role of Arg97 in Heat shock protein 90 N-terminal domain from the parasite Leishmania braziliensis through site-directed mutagenesis on the human counterpart.

In Brazil, the mucocutaneous form of leishmaniasis, caused by the parasite Leishmania braziliensis, is a widespread and very challenging disease responsible for disfiguration and, in the most severe cases, death. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone playing a pivotal role in the folding process of client proteins, and therefore its activity is fundamental for cell survival and proliferation. Since the chaperone activity requires ATP hydrolysis, molecules able to occupy the ATP binding pocket in the protein N-terminal domain (NTD) act as Hsp90 inhibitors. The development of selective molecules targeting the ATPase site of protozoan Hsp90 is tricky for the high homology with the human Hsp90 NTD (hNTD). Notably, only the human Lys112 is replaced by Arg97 in the L. braziliensis enzyme. Recently, this difference has been probed to design selective inhibitors targeting parasite Hsp90s. Here, a reliable protocol for expression and purification of LbHsp90-NTD (LbNTD) was developed but its structural characterization was unsuccessful. The role of Arg97 in LbNTD was hence probed by means of the "leishmanized" K112R variant of hNTDα. To deeply investigate the role of this residue, also the hNTDα K112A variant was generated. Structural studies performed on hNTDα and its variants using various ADP and ATP analogues and cAMP revealed that this residue is not crucial for nucleotide binding. This finding strongly suggests that Arg97 in LbNTD and more generally the conserved arginine residue in parasite Hsp90s are not exploitable for the development of selective inhibitors.

Non-coding RNAs in Host-Pathogen Interactions: Subversion of Mammalian Cell Functions by Protozoan Parasites.

Pathogens have evolved mechanisms to modulate host cell functions and avoid recognition and destruction by the host damage response. For many years, researchers have focused on proteins as the main effectors used by pathogens to hijack host cell pathways, but only recently with the development of deep RNA sequencing these molecules were brought to light as key players in infectious diseases. Protozoan parasites such as those from the genera Plasmodium, Toxoplasma, Leishmania, and Trypanosoma cause life-threatening diseases and are responsible for 1000s of deaths worldwide every year. Some of these parasites replicate intracellularly when infecting mammalian hosts, whereas others can survive and replicate extracellularly in the bloodstream. Each of these parasites uses specific evasion mechanisms to avoid being killed by the host defense system. An increasing number of studies have shown that these pathogens can transfer non-coding RNA molecules to the host cells to modulate their functions. This transference usually happens via extracellular vesicles, which are small membrane vesicles secreted by the microorganism. In this mini-review we will combine published work regarding several protozoan parasites that were shown to use non-coding RNAs in inter-kingdom communication and briefly discuss future perspectives in the field.

Desempenho e estudo de depleção residual em frangos de corte infectados com eimerias e medicados com diclazuril

A avicultura brasileira atualmente ocupa o terceiro lugar, com uma produção anual de aproximadamente, 10,9 milhões de toneladas de carne de frango. Contudo, severas perdas econômicas são relatadas, devido à coccidiose em granjas de frangos de corte, matrizes e postura. As Eimerias são classificadas como protozoários, sendo que os mesmos se multiplicam nas células intestinais diminuindo a absorção de nutrientes, levando à desidratação, perda de sangue e susceptibilidade para infecção por outros micro-organismos. Com o desenvolvimento da pesquisa objetivou-se determinar os índices de produtividade zootécnica (ganho de peso, consumo de ração, conversão alimentar, taxa de mortalidade e índice de eficiência produtiva) bem como, mensurar o nível residual do Diclazuril nos tecidos de frangos de corte, comparando com os padrões internacionais de Limites Máximos de Resíduos determinados pelo Codex Alimentarius. Para a realização do estudo, utilizou-se 624 frangos de corte, onde metade do grupo foi inoculado experimentalmente com E. acervulina, E. maxima e E. tenella. O estudo foi composto por grupos tratados e não tratados com diclazuril. O uso do diclazuril expressou efeito positivo, no desempenho zootécnico das aves inoculadas artificialmente; a análise residual do medicamento apresentou um período de carência zero, sendo considerada segura para alimentação humana a carne de frangos medicados com Diclazuril.(AU)

The Brazilian aviculture currently ranks third place with an annual production of approximately 10,9 million tons of chicken meat. However, severe economic losses are reported, due to coccidiosis in broiler chicken farms, breeders and posture. The Eimerias are classified as protozoa. They multiply in the intestinal cells decreasing the absorption of nutrients, leading to dehydration, blood loss and susceptibility to infection by other microorganisms. The development of research aimed to determine the levels of zootechnical productivity (weight gain, feed intake, feed conversion, mortality rate and productive efficiency index) as well measure the residual leveI of Diclazuril in broiler chicken tissues, compared to international standards for Maximum Residue limits established by the Codex Alimentarius. To conduct the study, we used 624 broilers, when half of the group had experimentally inoculated a mix of Eimerias: E. acervulina, E. maxima and E. tenella. The study consisted of treated and untreated groups with diclazuril. The use of diclazuril expressed positive effect on the zootechnical performance of artificially inoculated birds. The residual analysis of the drug had a zero waiting period, and is considered safe for human consumption the meat of chickens treated with diclazuril.(AU)

Desempenho e estudo de depleção residual em frangos de corte infectados com eimerias e medicados com diclazuril / Performance and residual depletion study in chickens infected with eimerias and medicated with diclazuril

A avicultura brasileira atualmente ocupa o terceiro lugar, com uma produção anual de aproximadamente, 10,9 milhões de toneladas de carne de frango. Contudo, severas perdas econômicas são relatadas, devido à coccidiose em granjas de frangos de corte, matrizes e postura. As Eimerias são classificadas como protozoários, sendo que os mesmos se multiplicam nas células intestinais diminuindo a absorção de nutrientes, levando à desidratação, perda de sangue e susceptibilidade para infecção por outros micro-organismos. Com o desenvolvimento da pesquisa objetivou-se determinar os índices de produtividade zootécnica (ganho de peso, consumo de ração, conversão alimentar, taxa de mortalidade e índice de eficiência produtiva) bem como, mensurar o nível residual do Diclazuril nos tecidos de frangos de corte, comparando com os padrões internacionais de Limites Máximos de Resíduos determinados pelo Codex Alimentarius. Para a realização do estudo, utilizou-se 624 frangos de corte, onde metade do grupo foi inoculado experimentalmente com E. acervulina, E. maxima e E. tenella. O estudo foi composto por grupos tratados e não tratados com diclazuril. O uso do diclazuril expressou efeito positivo, no desempenho zootécnico das aves inoculadas artificialmente; a análise residual do medicamento apresentou um período de carência zero, sendo considerada segura para alimentação humana a carne de frangos medicados com Diclazuril.

Infection of Human Monocytes with Leishmania infantum Strains Induces a Downmodulated Response when Compared with Infection with Leishmania braziliensis.

Human infection with different species of Leishmania leads to distinct clinical manifestations, ranging from relatively mild cutaneous (Leishmania braziliensis) to severe visceral (Leishmania infantum) forms of leishmaniasis. Here, we asked whether in vitro infection of human monocytes by Leishmania strains responsible for distinct clinical manifestations leads to early changes in immunological characteristics and ability of the host cells to control Leishmania. We evaluated the expression of toll-like receptors and MHC class II molecules, cytokines, and Leishmania control by human monocytes following short-term infection with L. braziliensis (M2904), a reference strain of L. infantum (BH46), and a wild strain of L. infantum (wild). The induction of TLR2, TLR9, and HLA-DR were all lower in L. infantum when compared with L. braziliensis-infected cells. Moreover, L. infantum-infected monocytes (both strains) produced lower TNF-alpha and a lower TNF-alpha/IL-10 ratio, resulting in a weaker inflammatory profile and a 100-fold less effective control of Leishmania than cells infected with L. braziliensis. Our results show that L. infantum strains fail to induce a strong inflammatory response, less activation, and less control of Leishmania from human monocytes, when compared with that induced by L. braziliensis infection. This functional profile may help explain the distinct clinical course observed in patients infected with the different Leishmania species.