Anti-Leishmania amazonensis activity of the marine sponge Dercitus (Stoeba) latex (Porifera) from São Pedro and São Paulo Archipelago, Pernambuco, Brazil.

The search for new therapeutic strategies for leishmaniasis treatment is essential due to the side effects of available drugs and the increasing incidence of resistance to them. Marine sponges use chemical compounds as a defense mechanism, and several of them present interesting pharmacological properties. The aim of this study was to evaluate the in vitro activity of the aqueous extract of the marine sponge Dercitus (Stoeba) latex against Leishmania amazonensis. MIC and toxicity against mammal cells were evaluated through broth microdilution assays. Transmission electron microscopy analysis was performed to assess possible effects on L. amazonensis ultrastructure. Arginase and proteolytic activities were measured by spectrometric methodologies. The extract of Dercitus (Stoeba) latex displayed antileishmanial activity and moderate toxicity against peritonial macrophages. Ultrastructural changes were observed after the growth of L. amazonensis promastigotes in the presence of the extract at 150 µg.ml-1 (IC50), mainly on acidocalcysomes. The extract was able to inhibit the activity of arginase and serine proteases. This study shows that Dercitus (Stoeba) latex aqueous extract may be a novel potential source of protozoa protease inhibitors and drugs that are less toxic to be used in the treatment of L. amazonensis infections.

Lopinavir and Nelfinavir Induce the Accumulation of Crystalloid Lipid Inclusions within the Reservosomes of Trypanosoma cruzi and Inhibit Both Aspartyl-Type Peptidase and Cruzipain Activities Detected in These Crucial Organelles.

Several research groups have explored the repositioning of human immunodeficiency virus aspartyl peptidase inhibitors (HIV-PIs) on opportunistic infections caused by bacteria, fungi and protozoa. In Trypanosoma cruzi, HIV-PIs have a high impact on parasite viability, and one of the main alterations promoted by this treatment is the imbalance in the parasite's lipid metabolism. However, the reasons behind this phenomenon are unknown. In the present work, we observed by transmission electron microscopy (TEM) that the treatment of T. cruzi epimastigotes with the HIV-PIs lopinavir and nelfinavir induced a huge accumulation of crystalloid-shaped lipids within the reservosomes, most of them deforming these key organelles. As previously reported, those structures are characteristic of lipid inclusions formed mostly of cholesterol and cholesterol-esters. The fractionation of nontreated epimastigotes generated two distinct fractions enriched in reservosomes: one mostly composed of lipid inclusion-containing reservosomes (Fraction B1) and one where lipid inclusions were much less abundant (Fraction B2). Interestingly, the extract of Fraction B2 presented enzymatic activity related to aspartyl-type peptidases 3.5 times higher than that found in the extract obtained from Fraction B1. The cleavage of cathepsin D substrate by this class of peptidases was strongly impaired by pepstatin A, a prototypical aspartyl PI, and the HIV-PIs lopinavir and nelfinavir. In addition, both HIV-PIs also inhibited (to a lesser extent) the cruzipain activity present in reservosomes. Finally, our work provides new evidence concerning the presence and supposed participation of aspartyl peptidases in T. cruzi, even as it adds new information about the mechanisms behind the alterations promoted by lopinavir and nelfinavir in the protozoan.

Endocytosis and Exocytosis in Leishmania amazonensis Are Modulated by Bromoenol Lactone.

In the protozoan pathogen Leishmania, endocytosis, and exocytosis occur mainly in the small area of the flagellar pocket membrane, which makes this parasite an interesting model of strikingly polarized internalization and secretion. Moreover, little is known about vesicle recognition and fusion mechanisms, which are essential for both endo/exocytosis in this parasite. In other cell types, vesicle fusion events require the activity of phospholipase A2 (PLA2), including Ca2+-independent iPLA2 and soluble, Ca2+-dependent sPLA2. Here, we studied the role of bromoenol lactone (BEL) inhibition of endo/exocytosis in promastigotes of Leishmania amazonensis. PLA2 activities were assayed in intact parasites, in whole conditioned media, and in soluble and extracellular vesicles (EVs) conditioned media fractions. BEL did not affect the viability of promastigotes, but reduced the differentiation into metacyclic forms. Intact parasites and EVs had BEL-sensitive iPLA2 activity. BEL treatment reduced total EVs secretion, as evidenced by reduced total protein concentration, as well as its size distribution and vesicles in the flagellar pocket of treated parasites as observed by TEM. Membrane proteins, such as acid phosphatases and GP63, became concentrated in the cytoplasm, mainly in multivesicular tubules of the endocytic pathway. BEL also prevented the endocytosis of BSA, transferrin and ConA, with the accumulation of these markers in the flagellar pocket. These results suggested that the activity inhibited by BEL, which is one of the irreversible inhibitors of iPLA2, is required for both endocytosis and exocytosis in promastigotes of L. amazonensis.

Trypanosoma cruzi: cell surface dynamics in trypomastigotes of different strains.

Capping and shedding of ectodomains in Trypanosoma cruzi may be triggered by different ligands. Here, we analysed the mobility and shedding of cell surface components of living trypomastigotes of the Y strain and the CL Brener clone in the presence of poly-L-lysine, cationized ferritin (CF) and Concanavalin A (Con A). Poly-L-lysine and CF caused intense shedding in Y strain parasites. Shedding was less intense in CL Brener trypomastigotes, and approximately 10% of these parasites did not show any decrease in poly L-lysine or CF labelling. Binding of Con A induced low-intensity shedding in Y strain and redistribution of Con A-binding sites in CL Brener parasites. Trypomastigotes of the Y strain showed intense labelling with anti-〈-galactosyl antibodies, resulting in the lysis of approximately 30% of their population, in contrast with what was observed in CL Brener parasites. Incubation with Con A and CF protected trypomastigotes of the Y strain from lysis by anti-αGal. The last treatment did not interfere with the survival of the CL Brener parasites. This study corroborates with the idea that a ligand can differentially modulate the cell surface of T. cruzi, depending on the strain used, resulting in variable immune system responses and recognition by host cells.

LPL protein in Chronic Lymphocytic Leukaemia have different origins in Mutated and Unmutated patients. Advances for a new prognostic marker in CLL.

Lipoprotein lipase (LPL) mRNA expression in chronic lymphocytic leukaemia (CLL) is associated with an unmutated immunoglobulin profile and poor clinical outcome. We evaluated the subcellular localization of LPL protein in CLL cells that did or did not express LPL mRNA. Our results show that LPL protein is differently located in CLL cells depending on whether it is incorporated from the extracellular medium in mutated CLL or generated de novo by leukaemic cells of unmutated patients. The specific quantification of endogenous LPL protein correlates with mRNA expression levels and mutational IGHV status, suggesting LPL protein as a possible reliable prognostic marker in CLL.

The in vitro antileishmanial activity of essential oil from Aloysia gratissima and guaiol, its major sesquiterpene against Leishmania amazonensis.

Leishmaniases is a tropical disease caused by protozoa of the genus Leishmania for which the current treatment is expensive, besides increasing reports of parasite resistance. This study investigated the anti-Leishmania amazonensis activity of the essential oil from Aloysia gratissima (AgEO) and guaiol, the major sesquiterpene constituent in the oil. Our results showed that AgEO killed promastigotes and intracellular amastigotes at an IC50 of 25 and 0·16 µg mL-1, respectively, while guaiol killed amastigotes at an IC50 of 0·01 µg mL-1. Both AgEO and guaiol were safe for macrophages up to 100 µg mL-1, as evaluated by the dehydrogenase activity, membrane integrity and phagocytic capacity. AgEO and guaiol did not induce nitrite oxide (NO) in resting macrophages and inhibited the production of NO in lipopolysaccharide-stimulated macrophages. The ultrastructural analysis suggested that AgEO and guaiol act directly on parasites, affecting promastigotes kinetoplast, mitochondrial matrix and plasma membrane. Together, these results pointed out that AgEO and guaiol could be promising candidates to develop anti-Leishmania drugs.

Triatoma infestans relies on salivary lysophosphatidylcholine to enhance Trypanosoma cruzi transmission.

Triatoma infestans is a mandatory haematophagous vector of Chagas disease in Brazil. Despite a large number of studies on the anti-haemostatic molecules present in its saliva, the role of its salivary components on parasite transmission is poorly understood. Here, we show that the bioactive lipid molecule, lysophosphatidylcholine (LPC), is present in the salivary gland of T. infestans. We characterized the lipid profiles of each unit of the T. infestans salivary gland. We noticed that LPC is present in the three units of the salivary gland and that the insect feeding state does not influence its proportion. T. infestans saliva and LPC can enhance T. cruzi transmission to mice by dramatically altering the profile of inflammatory cells at the site of inoculation on mouse skin, facilitating the transmission of T. cruzi to the vertebrate host. Consequently, the mortality curves of either saliva- or LPC-injected mice display significant higher mortality rates than the control. Altogether, these results implicate LPC as one of key salivary molecule involved in Chagas disease transmission.

Hyicin 4244, the first sactibiotic described in staphylococci, exhibits an anti-staphylococcal biofilm activity.

Hyicin 4244 is a small antimicrobial peptide with a broad spectrum of activity that was found in the culture supernatant of Staphylococcus hyicus 4244, the genome of which was then sequenced. The bacteriocin gene cluster (hyiSABCDEFG) was mined from its single chromosome and exhibited a genetic organization similar to that of subtilosin A. All genes involved in hyicin 4244 biosynthesis proved to be transcribed and encode proteins that share at least 42% similarity to proteins encoded by the subtilosin A gene cluster. Due to its resemblance to subtilosin A and the presence of three thioether bonds in its structure, hyicin 4244 is assumed to be a 35-amino acid circular sactibiotic, the first to be described in staphylococci. Hyicin 4244 inhibited 14 staphylococcal isolates from either human infections or bovine mastitis, all biofilm formers. Hyicin 4244 significantly reduced the number of colony-forming units (CFU) and the biofilm formation by two strong biofilm-forming strains randomly chosen as representatives of the strains involved in human infections and bovine mastitis. It also reduced the proliferation and viability of sessile cells in established biofilms. Therefore, hyicin 4244 proved not only to prevent biofilm formation by planktonic cells, but also to penetrate the biofilm matrix in vitro, exerting bactericidal activity against staphylococcal sessile cells. This bacteriocin has the potential to become an alternative antimicrobial for either prevention or treatment of biofilm-related infections caused by different staphylococcal species.

In vitro anti-MRSA activity of Couroupita guianensis extract and its component Tryptanthrin.

Couroupita guianensis is known in Brazil as 'Abricó-de-Macaco' and it has some attributes such as: antihypertensive, analgesic and anti-inflammatory activities. This study evaluated the antimicrobial activity of ethanolic extract and fractions of C. guianensis flowers and isolation of bioactive component. These extracts and fractions were subjected to agar diffusion, MIC, TLC and bioautography to bacteria, filamentous fungi and yeasts. Among the fractions of EtOH extract, the DCM fraction was the most active, particularly against Methicillin-resistant Staphylococcus aureus (MRSA) with MIC of 156 µg/mL. The active compound in this fraction was identified as Tryptanthrin, which showed promising antibacterial activity for MRSA showing MIC of 62.5 µg/mL. Ultrastructural analysis of MRSA incubated in the presence of Tryptanthrin by transmission electron microscope showed significant alterations in the cellular structure. Cytotoxicity tests demonstrated that DCM fraction and Tryptanthrin showed low toxicity, which makes it a promising candidate for alternative therapies to control and combat diseases.

Antiproliferative and ultrastructural effects of phenethylamine derivatives on promastigotes and amastigotes of Leishmania (Leishmania) infantum chagasi.

Leishmania (Leishmania) infantum chagasi is one of the agents that cause visceral leishmaniasis. This disease occurs more frequently in third world countries, such as Brazil. The treatment is arduous, and is dependent on just a few drugs like the antimonial derivatives and amphotericin B. Moreover, these drugs are not only expensive, but they can also cause severe side effects and require long-term treatment. Therefore, it is very important to find new compounds that are effective against leishmaniasis. In the present work we evaluated a new group of synthetic amides against the promastigote and amastigote forms of L. infantum chagasi. The results showed that one of these amides in particular, presented very effective activity against the promastigotes and amastigotes of L. infantum chagasi at low concentrations and it also presented low toxicity for mammal cells, which makes this synthetic amide a promising drug for combating leishmaniasis.

Phylogenetic and morphological characterization of trypanosomes from Brazilian armoured catfishes and leeches reveal high species diversity, mixed infections and a new fish trypanosome species.

BACKGROUND: Several Trypanosoma species transmitted by leeches infect marine and freshwater fish worldwide. To date, all South American fish trypanosome species identified have been based on unreliable morphological parameters. We recently isolated and cultured trypanosomes from the Brazilian armoured catfishes Hypostomus luetkeni and H. affinis. Here, we report the first phylogenetic analyses of South American (Brazilian) trypanosomes isolated from fish, and from leeches removed from these fish. We also analysed morphologically and morphometrically the different forms of fish, leech and cultured trypanosomes. METHODS: V7V8 SSU rRNA and gGAPDH sequences were used for phylogenetic analysis of Brazilian fish and leech trypanosomes. Trypanosomes from cultures, fish blood and leech samples were also characterized morphologically and morphometrically by light and electron microscopy. RESULTS: In blood smears from fish high trypanosome prevalence (90-100 %) and parasitemia (0.9-1.0x10(2)) were observed. Phylogenetic relationships using SSU rRNA and gGAPDH showed that, despite relevant sequence divergence, all Brazilian fish (and derived cultures) and leech trypanosomes clustered together into a single clade. The Brazilian clade clustered with European, North American and African fish trypanosomes. Based on sequence analysis, we uncovered a new species of Brazilian fish trypanosome, Trypanosoma abeli n. sp. Trypanosoma abeli cultures contained pleomorphic epimastigotes, small trypomastigotes and rare sphaeromastigotes. Ultrastructural features of T. abeli included a cytostome-cytopharynx complex in epi- and trypomastigotes, a compact rod-like kinetoplast, lysosome-related organelles (LROs) and multivesicular bodies. Trypanosomes found in fish blood smears and leech samples were highly pleomorphic, in agreement with sequence data suggesting that catfishes and leeches often have mixed trypanosome infections. CONCLUSIONS: Trypanosoma abeli n. sp. is the first trypanosome from South American fishes isolated in culture, positioned in phylogenetic trees and characterized at the ultrastructural level. Trypanosoma abeli n. sp. is highly prevalent in H. luetkeni and H. affinis armoured catfish from the Atlantic Forest biome, and in other catfish species from the Amazon and the Pantanal. Sequencing data suggested that Brazilian catfish often have mixed trypanosome infections, highlighting the importance of molecular characterization to identify trypanosome species in fishes and leeches.

Phenotypic and genotypic characterization of biofilm formation in Staphylococcus haemolyticus.

Staphylococcus haemolyticus is one of the most frequently isolated coagulase-negative staphylococci. The ability to produce biofilm has contributed to its emergence as a nosocomial pathogen. In this study, some growth conditions were tested to determine their influence on biofilm formation. Brain-heart infusion (BHI) broth containing glucose was used to screen 64 clinical strains. A strong biofilm producer strain showed cells surrounded by a thick layer of extracellular matrix. The presence of atlE, fbp, bap, and icaA genes was analyzed. We concluded that S. haemolyticus biofilm production can be increased with cells grown in BHI, and highlighted that it could be an ica-independent process.

Crovirin, a snake venom cysteine-rich secretory protein (CRISP) with promising activity against Trypanosomes and Leishmania.

BACKGROUND: The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania. METHODOLOGY/PRINCIPAL FINDINGS: Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10-2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells. CONCLUSIONS: This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.

Transovum transmission of trypanosomatid cysts in the Milkweed bug, Oncopeltus fasciatus.

Leptomonas wallacei is a trypanosomatid that develops promastigotes and cystic forms in the gut of the hemipteran insect Oncopeltus fasciatus. Insect trypanosomatids are thought to be solely transmitted from one host to another through the ingestion of parasite-contaminated feces. However, here we show that L. wallacei cysts present on the eggshells of eggs laid by O. fasciatus can also act as infective forms that are transmitted to the insect offspring. Newly hatched O. faciatus nymphs are parasite-free, but some of them become contaminated with L. wallacei after feeding on eggshell remnants. The present study is the first report of transovum transmission of a trypanosomatid, a process that may have a relevant role in parasite's within-host population dynamics.

Golgi UDP-GlcNAc:polypeptide O-α-N-Acetyl-d-glucosaminyltransferase 2 (TcOGNT2) regulates trypomastigote production and function in Trypanosoma cruzi.

All life cycle stages of the protozoan parasite Trypanosoma cruzi are enveloped by mucin-like glycoproteins which, despite major changes in their polypeptide cores, are extensively and similarly O-glycosylated. O-Glycan biosynthesis is initiated by the addition of αGlcNAc to Thr in a reaction catalyzed by Golgi UDP-GlcNAc:polypeptide O-α-N-acetyl-d-glucosaminyltransferases (ppαGlcNAcTs), which are encoded by TcOGNT1 and TcOGNT2. We now directly show that TcOGNT2 is associated with the Golgi apparatus of the epimastigote stage and is markedly downregulated in both differentiated metacyclic trypomastigotes (MCTs) and cell culture-derived trypomastigotes (TCTs). The significance of downregulation was examined by forced continued expression of TcOGNT2, which resulted in a substantial increase of TcOGNT2 protein levels but only modestly increased ppαGlcNAcT activity in extracts and altered cell surface glycosylation in TCTs. Constitutive TcOGNT2 overexpression had no discernible effect on proliferating epimastigotes but negatively affected production of both types of trypomastigotes. MCTs differentiated from epimastigotes at a low frequency, though they were apparently normal based on morphological and biochemical criteria. However, these MCTs exhibited an impaired ability to produce amastigotes and TCTs in cell culture monolayers, most likely due to a reduced infection frequency. Remarkably, inhibition of MCT production did not depend on TcOGNT2 catalytic activity, whereas TCT production was inhibited only by active TcOGNT2. These findings indicate that TcOGNT2 downregulation is important for proper differentiation of MCTs and functioning of TCTs and that TcOGNT2 regulates these functions by using both catalytic and noncatalytic mechanisms.

Structural and functional analysis of a platelet-activating lysophosphatidylcholine of Trypanosoma cruzi.

BACKGROUND: Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF. CONCLUSIONS/SIGNIFICANCE: Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynthesis could eventually be exploited as a potential target for new therapeutic interventions.

Trypanosoma cruzi-secreted vesicles have acid and alkaline phosphatase activities capable of increasing parasite adhesion and infection.

Trypanosoma cruzi virulence factors include molecules expressed on the cell surface as well as those secreted or shed into the extracellular medium. Phosphatase activities modulate different aspects of T. cruzi infection, although no studies to date addressed the presence and activity of phosphatases in vesicles secreted by this parasite. Here, we characterized acidic and alkaline secreted phosphatase activities of human-infective trypomastigote forms of T. cruzi from the Y strain and the CL-Brener clone. These are widely studied T. cruzi strains that represent "opposite ends of the spectrum" regarding both in vitro and in vivo behavior. Ecto-phosphatase activities were determined in live parasites, and secreted phosphatase activities were analyzed in soluble protein (SP) and vesicular membrane fractions (VFs) of parasite-conditioned medium. Our analysis using different phosphatase inhibitors strongly suggests that vesicles secreted by Y strain (VF(Y)) and CL-Brener (VF(CLB)) trypomastigotes are derived mostly from the cell surface and from exosome secretion, respectively. Importantly, our results show that the acid phosphatase activities in vesicles secreted by trypomastigotes are largely responsible for the VF-induced increase in adhesion of Y strain parasites to host cells and also for the VF-induced increase in host cell infection by CL-Brener trypomastigotes.

Effect of the secretory leucocyte proteinase inhibitor (SLPI) on Candida albicans biological processes: a therapeutic alternative?

OBJECTIVES: The aim of this study was to evaluate the effect of SLPI on the growth and biological processes of Candida albicans. METHODS: Two C. albicans strains were used in this study, a clinical isolate resistant to fluconazole (PRI) and a reference strain ATCC 24433. The minimal inhibitory concentration (MIC) was determined according to the CLSI methodology. The influence of SLPI on secreted serine proteinase activities (SSP) was measured by the cleavage of specific substrate, and surface hydrophobicity was determined by the aqueous-hydrocarbon biphasic separation method. Flow cytometry was performed to investigate receptors for SLPI and variations in the cell wall mannoprotein expression. Interaction between yeast and epithelium was assessed using the MA-104 cells lineage. Ultrastructure was analyzed by transmission electron microscopy (TEM). RESULTS: MIC values were calculated as 18 and 18.9µM for the PRI and ATCC 24433, respectively. SSP activity was reduced by 48.8% by 18µM of SLPI and cell surface hydrophobicity increased by 11.1%. Flow cytometry suggest the existence of SLPI binding sites on the surface of the yeast. Results showed a reduction in the expression of mannoproteins in 20.8% by the cells treated with 80µM of SLPI, and 18µM reduced the adhesion of yeasts to mammalian cells in 60.1%. TEM revealed ultrastructural changes in cells treated with 80µM of SLPI, such as the presence of membrane-like structures within the cytoplasm. CONCLUSIONS: SLPI exerts a significant influence on C. albicans viability and biological processes. Considering its constitutive and physiologic features, SLPI may become a promising tool for the development of new methodologies for the treatment and control of candidiasis.

Gene expression changes induced by Trypanosoma cruzi shed microvesicles in mammalian host cells: relevance of tRNA-derived halves.

At present, noncoding small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, canonical small RNA pathways seem to be lost or excessively simplified in some unicellular organisms including Trypanosoma cruzi which lack functional RNAi pathways. Recently, we reported the presence of alternate small RNA pathways in T. cruzi mainly represented by homogeneous populations of tRNA- and rRNA-derived small RNAs, which are secreted to the extracellular medium included in extracellular vesicles. Extracellular vesicle cargo could be delivered to other parasites and to mammalian susceptible cells promoting metacyclogenesis and conferring susceptibility to infection, respectively. Here we analyzed the changes in gene expression of host HeLa cells induced by extracellular vesicles from T. cruzi. As assessed by microarray assays a large set of genes in HeLa cells were differentially expressed upon incorporation of T. cruzi-derived extracellular vesicles. The elicited response modified mainly host cell cytoskeleton, extracellular matrix, and immune responses pathways. Some genes were also modified by the most abundant tRNA-derived small RNAs included in extracellular vesicles. These data suggest that microvesicles secreted by T. cruzi could be relevant players in early events of the T. cruzi host cell interplay.

Isolation and in vitro maintenance of trypanosomes from naturally infected and commercially important Brazilian fish.

Fish trypanosomes are widely distributed in commercially important fish, with high prevalence in some Brazilian species. This study provides the first record of the isolation and in vitro maintenance of trypanosomes from Brazilian fish. We produced 49 trypanosome isolates from naturally infected catfish (Hypostomus affinis and Hypostomus luetkeni), using 9 different culture media (out of 31 tested). Trypanosomes were maintained in culture for at least 15 mo and were successfully cryopreserved. Culture forms-epimastigotes and short trypomastigotes-were capable of dividing in vitro. Our study is an important step in the investigation of ultrastructure, taxonomy, and phylogeny of trypanosomes from commercially important Brazilian fish.