Trypanosoma cruzi pathogenicity involves virulence factor expression and upregulation of bioenergetic and biosynthetic pathways.

The molecular repertoire of Trypanosoma cruzi effects its virulence and impacts the clinical course of the resulting Chagas disease. This study aimed to determine the mechanism underlying the pathogenicity of T. cruzi. Two T. cruzi cell lines (C8C3hvir and C8C3lvir), obtained from the clone H510 C8C3 and exhibiting different virulence phenotypes, were used to evaluate the parasite's infectivity in mice. The organ parasite load was analysed by qPCR. The proteomes of both T. cruzi cell lines were compared using nLC-MS/MS. Cruzipain (Czp), complement regulatory protein (CRP), trans-sialidase (TS), Tc-85, and sialylated epitope expression levels were evaluated by immunoblotting. High-virulence C8C3hvir was highly infectious in mice and demonstrated three to five times higher infectivity in mouse myocardial cells than low-virulence C8C3lvir. qPCR revealed higher parasite loads in organs of acute as well as chronically C8C3hvir-infected mice than in those of C8C3lvir-infected mice. Comparative quantitative proteomics revealed that 390 of 1547 identified proteins were differentially regulated in C8C3hvir with respect to C8C3lvir. Amongst these, 174 proteins were upregulated in C8C3hvir and 216 were downregulated in C8C3lvir. The upregulated proteins in C8C3hvir were associated with the tricarboxylic acid cycle, ribosomal proteins, and redoxins. Higher levels of Czp, CRP, TS, Tc-85, and sialylated epitopes were expressed in C8C3hvir than in C8C3lvir. Thus, T. cruzi virulence may be related to virulence factor expression as well as upregulation of bioenergetic and biosynthetic pathways proteins.

In Silico Characterization of Calcineurin from Pathogenic Obligate Intracellular Trypanosomatids: Potential New Biological Roles.

Calcineurin (CaN) is present in all eukaryotic cells, including intracellular trypanosomatid parasites such as Trypanosoma cruzi (Tc) and Leishmania spp. (Lspp). In this study, we performed an in silico analysis of the CaN subunits, comparing them with the human (Hs) and looking their structure, post-translational mechanisms, subcellular distribution, interactors, and secretion potential. The differences in the structure of the domains suggest the existence of regulatory mechanisms and differential activity between these protozoa. Regulatory subunits are partially conserved, showing differences in their Ca2+-binding domains and myristoylation potential compared with human CaN. The subcellular distribution reveals that the catalytic subunits TcCaNA1, TcCaNA2, LsppCaNA1, LsppCaNA1_var, and LsppCaNA2 associate preferentially with the plasma membrane compared with the cytoplasmic location of HsCaNAα. For regulatory subunits, HsCaNB-1 and LsppCaNB associate preferentially with the nucleus and cytoplasm, and TcCaNB with chloroplast and cytoplasm. Calpain cleavage sites on CaNA suggest differential processing. CaNA and CaNB of these trypanosomatids have the potential to be secreted and could play a role in remote communication. Therefore, this background can be used to develop new drugs for protozoan pathogens that cause neglected disease.

Demodex folliculorum (Trombidiformes: Demodicidae) and Demodex brevis Prevalence in an Extreme Environment of Chile.

The prevalence of mites of the genus Demodex and their associations with host age, gender, workplace, and comorbid skin and ocular conditions were studied in participants in Antofagasta, Chile, which is in a region with an extreme environment. We examined 680 participants aged 18-88 yr using standardized surface skin biopsies. Among them, Demodex had a prevalence of 13.5 % (95% confidence interval, 10.88-16.17). A slightly higher prevalence was observed in males (51.1%; 95% confidence interval, 40.9-61.3; nonsignificant) and participants in the 69-88 yr age group (50.0%; 95% confidence interval, 23.8-76.2; P < 0.05). Regarding the species involved, Demodex folliculorum was found in 89.1% (CI 82.7-95.5) of cases, while D. brevis was found in the remaining 10.9% of cases. A higher prevalence (25.0% CI 16.1-33.91) was observed in participants who worked indoors in generally enclosed and dust-rich environments (such as theaters, libraries, and administrative offices). There was also a strong association between Demodex prevalence and conjunctival hyperemia, with 35.9% (95% confidence interval, 9.1-35; OR 17.9) of the Demodex positive participants having this pathology compared to 10.3% of the noninfested participants. In summary, the prevalence of Demodex in Antofagasta, Chile (13.5%) was toward the lower end of the range reported among other regions around the world. Environmental factors such as exposure to the sun (including ultraviolet rays) or environmental pollution may affect the mites. In addition, Demodex genetics (related to virulence) and the ocular or skin microbiota may positively or negatively influence infestation and pathology.

Arsenic trioxide-increased MDCK cells proliferation requires activator protein 1-mediated increase of the sodium/proton exchanger 1 activity.

The release of protons (H+) occurs via the Na+/H+ exchanger isoform 1 (NHE1) leading to a stable intracellular pH (pHi) in MDCK cells. Chronic intake of arsenic trioxide (ATO), in the drinking water, associated with higher morbidity and mortality in neoplastic tissues. ATO increased NHE1 expression and activity, resulting in intracellular alkalization and higher MDCK cells proliferation. Since the pro-proliferative transcription factor activator protein 1 (AP-1) gets activated by al alkaline intracellular pH, a phenomenon paralleled by higher NHEs activity, we asked whether ATO-increased MDCK cells proliferation involves AP-1-dependent NHE1 activation. Cells were exposed (48 h) to ATO (0.05 µmol/L), SR11302 (1 µmol/L, AP-1 inhibitor), HOE-694 (100 nmol/L, NHE1 inhibitor) and EIPA (50 µmol/L, NHE1/NHE3 inhibitor) in the presence of S3226 (10 µmol/L, NHE3 inhibitor), concanamycin A (0.1 µmol/L, V-ATPases inhibitor), and Schering (10 µmol/L, H+/K+-ATPase inhibitor). [3H]Thymidine incorporation, cell counting, wound healing assay, and AP-1 activity were determined. The pHi was measured in cells pre-loaded (10 min) with 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (12 mmol/L) and exposed to NH4Cl (20 mmol/L). Basal pHi and recovery rate (dpHi/dt), intracellular buffer capacity (ßi) and H+ flux (JH+) were determined. NHE1 protein abundance was measured by Western blotting and immunofluorescence. ATO increased the cell growth (1.5 fold), basal pHi (0.4 pHi units), dpHi/dt (1.8 fold), JH+ (1.4 fold), AP-1 activity and NHE1 protein abundance (1.3 fold). ATO also increased (1.5 fold) the nuclear/perinuclear NHE1 immunosignal. SR11302 and HOE-694 blocked ATO effects. Thus, ATO-increased proliferation resulted from AP-1-dependent NHE1 activation in MDCK cells.

Effect of DODAB Nano-Sized Cationic Bilayer Fragments against Leishmania amazonensis.

The dioctadecyldimethylammonium bromide (DODAB) is a double-chained cationic lipid with potent bactericide and fungistatic activities; however, its toxicity on protozoan parasites is still unknown. Here, we show the antileishmanial activity of DODAB nano-sized cationic bilayer fragments on stationary-phase promastigotes and amastigotes of Leishmania amazonensis, the causative agent of cutaneous leishmaniasis. Upon treatment with DODAB, we analyzed the parasite surface zeta-potential, parasite viability, cellular structural modifications, and intracellular proliferation. The DODAB cytotoxic effect was dose-dependent, with a median effective concentration (EC50) of 25 µM for both life-cycle stages, comparable to the reported data for bacteria and fungi. The treatment with DODAB changed the membrane zeta-potential from negative to positive, compromised the parasite's morphology, affected the cell size regulation, caused a loss of intracellular organelles, and probably dysregulated the plasma membrane permeability without membrane disruption. Moreover, the parasites that survived after treatment induced small parasitophorous vacuoles and failed to proliferate inside macrophages. In conclusion, DODAB displayed antileishmanial activity, and it remains to be elucidated how DODAB acts on the protozoan membrane. Understanding this mechanism can provide insights into the development of new parasite-control strategies.

Toxicological Characterization and Phospholipase D Activity of the Venom of the Spider Sicarius thomisoides.

Envenomation by Loxosceles spiders (Sicariidae family) has been thoroughly documented. However, little is known about the potential toxicity of members from the Sicarius genus. Only the venom of the Brazilian Sicarius ornatus spider has been toxicologically characterized. In Chile, the Sicarius thomisoides species is widely distributed in desert and semidesert environments, and it is not considered a dangerous spider for humans. This study aimed to characterize the potential toxicity of the Chilean S. thomisoides spider. To do so, specimens of S. thomisoides were captured in the Atacama Desert, the venom was extracted, and the protein concentration was determined. Additionally, the venoms were analyzed by electrophoresis and Western blotting using anti-recombinant L. laeta PLD1 serum. Phospholipase D enzymatic activity was assessed, and the hemolytic and cytotoxic effects were evaluated and compared with those of the L. laeta venom. The S. thomisoides venom was able to hydrolyze sphingomyelin as well as induce complement-dependent hemolysis and the loss of viability of skin fibroblasts with a dermonecrotic effect of the venom in rabbits. The venom of S. thomisoides showed intraspecific variations, with a similar protein pattern as that of L. laeta venom at 32-35 kDa, recognized by serum anti-LlPLD1. In this context, we can conclude that the venom of Sicarius thomisoides is similar to Loxosceles laeta in many aspects, and the dermonecrotic toxin present in their venom could cause severe harm to humans; thus, precautions are necessary to avoid exposure to their bite.

Staphylococcus sciuri Strain LCHXa is a Free-Living Lithium-Tolerant Bacterium Isolated from Salar de Atacama, Chile.

In addition to the industrial and biomedical applications of lithium, information on the tolerance of microorganisms to high Li concentrations in natural biological systems is limited. Strain LCHXa is a novel free-living Gram-positive, non-motile bacterium strain isolated from water samples taken at Laguna Chaxa, a non-industrial water body with the highest soluble Li content (33 mM LiCl) within the Salar de Atacama basin in northern Chile. Enrichment was conducted in Luria-Bertani (LB) medium supplemented with 1 M LiCl. Strain LCHXa was a Novobiocin-resistant and coagulase negative Staphylococcus. Phylogenetically, strain LCHXa belongs to the species Staphylococcus sciuri. Strain LCHXa grew optimally in LB medium at pH 6-8 and 37 °C, and it was able to sustain growth at molar Li concentrations at 2 M LiCl, with a decrease in the specific growth rate of 85%. Osmoregulation in strain LCHXa partially involves glycine betaine and glycerol as compatible solutes.

Haloterrigena sp. Strain SGH1, a Bacterioruberin-Rich, Perchlorate-Tolerant Halophilic Archaeon Isolated From Halite Microbial Communities, Atacama Desert, Chile.

An extreme halophilic archaeon, strain SGH1, is a novel microorganism isolated from endolithic microbial communities colonizing halites at Salar Grande, Atacama Desert, in northern Chile. Our study provides structural, biochemical, genomic, and physiological information on this new isolate living at the edge of the physical and chemical extremes at the Atacama Desert. SGH1 is a Gram-negative, red-pigmented, non-motile unicellular coccoid organism. Under the transmission electron microscope, strain SGH1 showed an abundant electro-dense material surrounding electron-lucent globular structures resembling gas vacuoles. Strain SGH1 showed a 16S rRNA gene sequence with a close phylogenetic relationship to the extreme halophilic archaea Haloterrigena turkmenica and Haloterrigena salina and has been denominated Haloterrigena sp. strain SGH1. Strain SGH1 grew at 20-40°C (optimum 37°C), at salinities between 15 and 30% (w/v) NaCl (optimum 25%) and growth was improved by addition of 50 mM KCl and 0.5% w/v casamino acids. Growth was severely restricted at salinities below 15% NaCl and cell lysis is avoided at a minimal 10% NaCl. Maximal concentrations of magnesium chloride and sodium or magnesium perchlorates that supported SGH1 growth were 0.5 and 0.15M, respectively. Haloterrigena sp. strain SGH1 accumulates bacterioruberin (BR), a C50 xanthophyll, as the major carotenoid. Total carotenoids in strain SGH1 amounted to nearly 400 µg BR per gram of dry biomass. Nearly 80% of total carotenoids accumulated as geometric isomers of BR: all-trans-BR (50%), 5-cis-BR (15%), 9-cis-BR (10%), 13-cis-BR (4%); other carotenoids were dehydrated derivatives of BR. Carotenogenesis in SGH1 was a reversible and salt-dependent process; transferring BR-rich cells grown in 25% (w/v) NaCl to 15% (w/v) NaCl medium resulted in depigmentation, and BR content was recovered after transference and growth of unpigmented cells to high salinity medium. Methanol extracts and purified BR isomers showed an 8-9-fold higher antioxidant activity than Trolox or ß-carotene. Both, plasma membrane integrity and mitochondrial membrane potential measurements under acute 18-h assays showed that purified BR isomers were non-toxic to cultured human THP-1 cells.

Observational cross-sectional study of Trichomonas tenax in patients with periodontal disease attending a Chilean university dental clinic.

BACKGROUND: The oral flagellated protozoan Trichomonas tenax has been associated with patients with periodontal disease. However, no recent studies have been conducted on the prevalence of T. tenax in Chile. The aim of this study was to determine the presence of T. tenax in patients with periodontal disease, admitted to the Dental Clinic of the University of Antofagasta, Chile, through Polymerase Chain Reaction (PCR) amplification of the beta-tubulin gene. METHODS: An observational, cross-sectional study was conducted on 50 patients diagnosed with periodontal disease, 20 of them with gingivitis and 30 with periodontitis. T. tenax was identified by PCR amplification of the beta-tubulin gene. Associations between the protozoan and periodontal disease or the presence of risk factors to establish T. tenax infection were determined using the chi-square test and binary logistic regression analysis. RESULTS: T. tenax was present in 28 out of 50 (56%) of patients with periodontal disease, and was more prevalent when associated with periodontitis (21 out of 30; 70%) than dental plaque-induced gingivitis (7 out of 20; 35%). Non-statistically-significant associations were observed between the presence of T. tenax and age, gender, smoking habit or diabetes. Statistically significant associations were observed between the presence of T. tenax and periodontal disease, and between T. tenax and the Periodontal Screening and Recording (PSR) index. CONCLUSION: T. tenax showed a high presence in patients with progressive states of periodontal diseases. Consequently, T. tenax detection is strongly recommended in patients with periodontal disease diagnosis and with a PSR index greater than 3.

Draft Genome Sequence of Staphylococcus sciuri Strain LCHXa, a Lithium-Tolerant Bacterium Isolated from Laguna Chaxa, Salar de Atacama, Chile.

A Gram-positive, coagulase-negative, novobiocin resistant, and lithium-tolerant bacterium was isolated from Salar de Atacama. Strain LCHXa is closely related to Staphylococcus sciuri. Its genome is 3,013,090 bp long and contains 2,551 predicted protein genes. We observed 58 genes associated with stress response and 17 genes linked to osmoregulation, mainly related to glycine betaine metabolism.

Involvement of Intracellular pH in Vascular Insulin Resistance.

The maintenance of the pH homeostasis is maintained by several mechanisms including the efflux of protons (H+) via membrane transporters expressed in almost all mammalian cells. Along these membrane transporters the sodium/H+ exchangers (NHEs), mainly NHE isoform 1 (NHE1), plays a key role in this phenomenon. NHE1 is under modulation by several environmental conditions (e.g. hyperglycaemia, protein kinase C activity) as well as hormones, including insulin. NHE1 activation causes intracellular alkalization in human endothelial cells leading to activation of the endothelial Nitric Oxide Synthase (eNOS) to generate NO. Intracellular alkalization is a phenomenon that also results in upregulation of the glucose transporter GLUT4 in cells that are responsive to insulin. A reduction in the removal of the extracellular D-glucose is seen in states of insulin resistance, such as in diabetes mellitus and obesity. Since insulin is a potent activator of eNOS in human endothelium, therefore causing vasodilation, and its vascular effect is reduced in insulin resistance it is likely that a defective signal to activate NHE1 in insulin target cells is expected. This phenomenon results in lower redistribution and activation of GLUT4 leading to reduced uptake of D-glucose and hyperglycaemia. The general concept of a role for NHE1, and perhaps other NHEs isoforms, in insulin resistance in the human vasculature is proposed.

Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders.

BACKGROUND: Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. METHODS: The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. RESULTS: We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. CONCLUSIONS: People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.

Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Background Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.

Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.(AU)

Phospholipase D from Loxosceles laeta Spider Venom Induces IL-6, IL-8, CXCL1/GRO-α, and CCL2/MCP-1 Production in Human Skin Fibroblasts and Stimulates Monocytes Migration.

Cutaneous loxoscelism envenomation by Loxosceles spiders is characterized by the development of a dermonecrotic lesion, strong inflammatory response, the production of pro-inflammatory mediators, and leukocyte migration to the bite site. The role of phospholipase D (PLD) from Loxosceles in the recruitment and migration of monocytes to the envenomation site has not yet been described. This study reports on the expression and production profiles of cytokines and chemokines in human skin fibroblasts treated with catalytically active and inactive recombinant PLDs from Loxosceles laeta (rLlPLD) and lipid inflammatory mediators ceramide 1-phosphate (C1P) and lysophosphatidic acid (LPA), and the evaluation of their roles in monocyte migration. Recombinant rLlPLD1 (active) and rLlPLD2 (inactive) isoforms induce interleukin (IL)-6, IL-8, CXCL1/GRO-α, and CCL2/monocyte chemoattractant protein-1 (MCP-1) expression and secretion in fibroblasts. Meanwhile, C1P and LPA only exhibited a minor effect on the expression and secretion of these cytokines and chemokines. Moreover, neutralization of both enzymes with anti-rLlPLD1 antibodies completely inhibited the secretion of these cytokines and chemokines. Importantly, conditioned media from fibroblasts, treated with rLlPLDs, stimulated the transmigration of THP-1 monocytes. Our data demonstrate the direct role of PLDs in chemotactic mediator synthesis for monocytes in human skin fibroblasts and indicate that inflammatory processes play an important role during loxoscelism.

Decreased cruzipain and gp85/trans-sialidase family protein expression contributes to loss of Trypanosoma cruzi trypomastigote virulence.

Two cell lines derived from a single Trypanosoma cruzi clone by long-term passaging generated a highly virulent (C8C3hvir) and a low virulent (C8C3lvir) cell line. The C8C3hvir cell line was highly infective and lethal to Balb/c mice, and the C8C3lvir cell line was three- to five-fold less infective to mouse cardiomyocytes than C8C3hvir. The highly virulent T. cruzi cell line abundantly expressed the major cysteine proteinase cruzipain (Czp), complement regulatory protein (CRP) and trans-sialidase (TS), all of which are known to act as virulence factors in this parasite. The in vitro invasion capacity and in vivo Balb/c mouse infectiveness of the highly virulent strain was strongly reduced by pre-treatment with antisense oligonucleotides targeting TS or CRP or with E64d. Based on these results, we conclude that decreased levels of TS, CRP and Czp expression could contribute to loss of T. cruzi trypomastigote virulence.

Draft Genome Sequence of a Bacillus Bacterium from the Atacama Desert Wetlands Metagenome.

We report here the draft genome sequence of a Bacillus bacterium isolated from the microflora of Nostoc colonies grown at the Andean wetlands in northern Chile. We consider this genome sequence to be a molecular tool for exploring microbial relationships and adaptation strategies to the prevailing extreme conditions at the Atacama Desert.

A cytoplasmic new catalytic subunit of calcineurin in Trypanosoma cruzi and its molecular and functional characterization.

Parasitological cure for Chagas disease is considered extremely difficult to achieve because of the lack of effective chemotherapeutic agents against Trypanosoma cruzi at different stages of infection. There are currently only two drugs available. These have several limitations and can produce serious side effects. Thus, new chemotherapeutic targets are much sought after. Among T. cruzi components involved in key processes such as parasite proliferation and host cell invasion, Ca(2+)-dependent molecules play an important role. Calcineurin (CaN) is one such molecule. In this study, we cloned a new isoform of the gene coding for CL strain catalytic subunit CaNA (TcCaNA2) and characterized it molecularly and functionally. There is one copy of the TcCaNA2 gene per haploid genome. It is constitutively transcribed in all T. cruzi developmental forms and is localized predominantly in the cytosol. In the parasite, TcCaNA2 is associated with CaNB. The recombinant protein TcCaNA2 has phosphatase activity that is enhanced by Mn(2+)/Ni(2+). The participation of TcCaNA2 in target cell invasion by metacyclic trypomastigotes was also demonstrated. Metacyclic forms with reduced TcCaNA2 expression following treatment with morpholino antisense oligonucleotides targeted to TcCaNA2 invaded HeLa cells at a lower rate than control parasites treated with morpholino sense oligonucleotides. Similarly, the decreased expression of TcCaNA2 following treatment with antisense morpholino oligonucleotides partially affected the replication of epimastigotes, although to a lesser extent than the decrease in expression following treatment with calcineurin inhibitors. Our findings suggest that the calcineurin activities of TcCaNA2/CaNB and TcCaNA/CaNB, which have distinct cellular localizations (the cytoplasm and the nucleus, respectively), may play a critical role at different stages of T. cruzi development, the former in host cell invasion and the latter in parasite multiplication.

Tryptophan and aspartic acid residues present in the glycerophosphoryl diester phosphodiesterase (GDPD) domain of the Loxosceles laeta phospholipase D are essential for substrate recognition.

It is known that the family of phospholipases D (PLD) from spiders of the genus Loxosceles, hydrolyze the substrates sphingomyelin and lisophosphatidylcholine, by their catalytic acid-base action which involves two histidines. However, little is known about the amino acids that participate on substrate recognition. In this study we identified highly conserved amino acids of the glycerophosphoryl diester phosphodiesterase (GDPD) domain of recombinant LlPLD1, which interact with the substrate sphingomyelin. The mutation of W256 to serine and D259 to glycine decreased significantly the sphingomyelinase and hemolytic activity when compared to wild type LlPLD1. The interaction of LlPLD1 with sphingomyelin was also strongly reduced in both mutants LlPLD1-W256S and LlPLD1-D259G. The results show the importance of these residues in the interaction of the protein with its substrate sphingomyelin in cell membranes.

Aromatic glycosyl disulfide derivatives: evaluation of their inhibitory activities against Trypanosoma cruzi.

Aromatic oligovalent glycosyl disulfides and some diglycosyl disulfides were tested against three different Trypanosoma cruzi strains. Di-(ß-D-galactopyranosyl-dithiomethylene) benzenes 2b and 4b proved to be the most active derivatives against all three strains of cell culture-derived trypomastigotes with IC50 values ranging from 4 to 11 µM at 37 °C. The inhibitory activities were maintained, although somewhat lowered, at a temperature of 4 °C as well. Three further derivatives displayed similar activities against at least one of the three strains. Low cytotoxicities of the active compounds, tested on confluent HeLa, Vero and peritoneal macrophage cell cultures, resulted in significantly higher selectivity indices (SI) than that of the reference drug benznidazole. Remarkably, several molecules of the tested panel strongly inhibited the parasite release from T. cruzi infected HeLa cell cultures suggesting an effect against the intracellular development of T. cruzi amastigotes as well.