A versatile 2A peptide-based strategy for ectopic expression and endogenous gene tagging in Trypanosoma cruzi.

Nearly all expression vectors currently available for Trypanosoma cruzi were conceived to produce a single primary transcript containing the genes of interest along with those that confer antibiotic resistance. However, since each messenger RNA (mRNA) matures separately, drug selection will only guarantee the expression of those derived from the selectable marker. Therefore, commonly a considerable fraction of the cells recovered after selection with these expression vectors, although resistant do not express the protein of interest. Consequently, in order to counteract this disadvantage, we developed vectors with an alternative arrangement in which the gene of interest and antibiotic resistance are fused sharing the same mRNA. To test this configuration, we included the coding sequence for the green fluorescent protein (mEGFP) linked to the one conferring neomycin resistance (Neo). Additionally, to allow for the production of two independent proteins the sequence for a Thosea asigna virus self-cleaving 2A peptide (T2A) was inserted in-between. Cells obtained with these vectors displayed higher mEGFP expression levels with more homogeneous transgenic parasite populations than those transfected with more conventional independent mRNA-based alternatives. Moreover, as determined by Western blot, 2A mediated fusion protein dissociation occurred with high efficiency in all parasite stages. In addition, these vectors could easily be transformed into endogenous tagging constructs that allowed the insertion, by ends-in homologous recombination, of a hemagglutinin tag (HA) fused to the actin gene. The use of 2A self-cleaving peptides in the context of single mRNA vectors represents an interesting strategy capable of improving ectopic transgene expression in T. cruzi as well as providing a simple alternative to more sophisticated methods, such as the one based on CRISPR/Cas9, for the endogenous labeling of genes.

Exploring the Pathophysiologic Cascade Leading to Osteoclastogenic Activation in Gaucher Disease Monocytes Generated via CRISPR/Cas9 Technology.

Gaucher disease (GD) is caused by biallelic pathogenic variants in the acid ß-glucosidase gene (GBA1), leading to a deficiency in the ß-glucocerebrosidase (GCase) enzyme activity resulting in the intracellular accumulation of sphingolipids. Skeletal alterations are one of the most disabling features in GD patients. Although both defective bone formation and increased bone resorption due to osteoblast and osteoclast dysfunction contribute to GD bone pathology, the molecular bases are not fully understood, and bone disease is not completely resolved with currently available specific therapies. For this reason, using editing technology, our group has developed a reliable, isogenic, and easy-to-handle cellular model of GD monocytes (GBAKO-THP1) to facilitate GD pathophysiology studies and high-throughput drug screenings. In this work, we further characterized the model showing an increase in proinflammatory cytokines (Interleukin-1ß and Tumor Necrosis Factor-α) release and activation of osteoclastogenesis. Furthermore, our data suggest that GD monocytes would display an increased osteoclastogenic potential, independent of their interaction with the GD microenvironment or other GD cells. Both proinflammatory cytokine production and osteoclastogenesis were restored at least, in part, by treating cells with the recombinant human GCase, a substrate synthase inhibitor, a pharmacological chaperone, and an anti-inflammatory compound. Besides confirming that this model would be suitable to perform high-throughput screening of therapeutic molecules that act via different mechanisms and on different phenotypic features, our data provided insights into the pathogenic cascade, leading to osteoclastogenesis exacerbation and its contribution to bone pathology in GD.

Anti-Trypanosoma cruzi Properties of Sesquiterpene Lactones Isolated from Stevia spp.: In Vitro and In Silico Studies.

Stevia species (Asteraceae) have been a rich source of terpenoid compounds, mainly sesquiterpene lactones, several of which show antiprotozoal activity. In the search for new trypanocidal compounds, S. satureiifolia var. satureiifolia and S. alpina were studied. Two sesquiterpene lactones, santhemoidin C and 2-oxo-8-deoxyligustrin, respectively, were isolated. These compounds were assessed in vitro against Trypanosoma cruzi stages, showing IC50 values of 11.80 and 4.98 on epimastigotes, 56.08 and 26.19 on trypomastigotes and 4.88 and 20.20 µM on amastigotes, respectively. Cytotoxicity was evaluated on Vero cells by the MTT assay. The effect of the compounds on trypanothyone reductase (TcTR), Trans-sialidase (TcTS) and the prolyl oligopeptidase of 80 kDa (Tc80) as potential molecular targets of T. cruzi was investigated. Santhemoidin C inhibited oligopeptidase activity when tested against recombinant Tc80 using a fluorometric assay, reaching an IC50 of 34.9 µM. Molecular docking was performed to study the interaction between santhemoidin C and the Tc80 protein, reaching high docking energy levels. Plasma membrane shedding and cytoplasmic vacuoles, resembling autophagosomes, were detected by transmission microscopy in parasites treated with santhemoidin C. Based on these results, santhemoidin C represents a promising candidate for further studies in the search for new molecules for the development of trypanocidal drugs.

Trypanosoma cruzi Induces B Cells That Regulate the CD4+ T Cell Response.

Trypanosoma cruzi infection induces a polyclonal B cell proliferative response characterized by maturation to plasma cells, excessive generation of germinal centers, and secretion of parasite-unrelated antibodies. Although traditionally reduced to the humoral response, several infectious and non-infectious models revealed that B lymphocytes could regulate and play crucial roles in cellular responses. Here, we analyze the trypomastigote-induced effect on B cells, their effects on CD4+ T cells, and their correlation with in vivo findings. The trypomastigotes were able to induce the proliferation and the production of IL-10 or IL-6 of naïve B cells in co-culture experiments. Also, we found that IL-10-producing B220lo cells were elicited in vivo. We also found up-regulated expression of FasL and PD-L1, proteins involved in apoptosis induction and inhibition of TCR signaling, and of BAFF and APRIL mRNAs, two B-cell growth factors. Interestingly, it was observed that IL-21, which plays a critical role in regulatory B cell differentiation, was significantly increased in B220+/IL-21+ in in vivo infections. This is striking since the secretion of IL-21 is associated with T helper follicular cells. Furthermore, trypomastigote-stimulated B-cell conditioned medium dramatically reduced the proliferation and increased the apoptotic rate on CD3/CD28 activated CD4+ T cells, suggesting the development of effective regulatory B cells. In this condition, CD4+ T cells showed a marked decrease in proliferation and viability with marginal IL-2 or IFNγ secretion, which is counterproductive with an efficient immune response against T. cruzi. Altogether, our results show that B lymphocytes stimulated with trypomastigotes adopt a particular phenotype that exerts a strong regulation of this T cell compartment by inducing apoptosis, arresting cell division, and affecting the developing of a proinflammatory response.

Parasite-host glycan interactions during Trypanosoma cruzi infection: trans-Sialidase rides the show.

Many important pathogen-host interactions rely on highly specific carbohydrate binding events. In the case of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, glycointeractions involving sialic acid (SA) residues are pivotal for parasite infectivity, escape from immune surveillance and pathogenesis. Though unable to synthesize SA de novo, T. cruzi displays a unique trans-Sialidase (TS) enzyme, which is able to cleave terminal SA residues from host donor glycoconjugates and transfer them onto parasite surface mucins, thus generating protective/adhesive structures. In addition, this parasite sheds TS into the bloodstream, as a way of modifying the surface SA signature, and thereby the signaling/functional properties of mammalian host target cells on its own advantage. Here, we discuss the pathogenic aspects of T. cruzi TS: its molecular adaptations, the multiplicity of interactions in which it is involved during infections, and the array of novel and appealing targets for intervention in Chagas disease provided by TS-remodeled sialoglycophenotypes.

Efficacy of pentosan polysulfate in in vitro models of lysosomal storage disorders: Fabry and Gaucher Disease.

Gaucher and Fabry diseases are the most prevalent sphingolipidoses. Chronic inflammation is activated in those disorders, which could play a role in pathogenesis. Significant degrees of amelioration occur in patients upon introduction of specific therapies; however, restoration to complete health status is not always achieved. The idea of an adjunctive therapy that targets inflammation may be a suitable option for patients. PPS is a mixture of semisynthetic sulfated polyanions that have been shown to have anti-inflammatory effects in mucopolysaccharidosis type I and II patients and animal models of type I, IIIA and VI. We hypothesized PPS could be a useful adjunctive therapy to inflammation for Gaucher and Fabry diseases. The objective of this work is to analyze the in vitro effect of PPS on inflammatory cytokines in cellular models of Gaucher and Fabry diseases, and to study its effect in Gaucher disease associated in vitro bone alterations. Cultures of peripheral blood mononuclear cells from Fabry and Gaucher patients were exposed to PPS. The secretion of proinflammatory cytokines was significantly reduced. Peripheral blood cells exposed to PPS from Gaucher patients revealed a reduced tendency to differentiate to osteoclasts. Osteoblasts and osteocytes cell lines were incubated with an inhibitor of glucocerebrosidase, and conditioned media was harvested in order to analyze if those cells secrete factors that induce osteoclastogenesis. Conditioned media from this cell cultures exposed to PPS produced lower numbers of osteoclasts. We could demonstrate PPS is an effective molecule to reduce the production of proinflammatory cytokines in in vitro models of Fabry and Gaucher diseases. Moreover, it was effective at ameliorating bone alterations of in vitro models of Gaucher disease. These results serve as preclinical supportive data to start clinical trials in human patients to analyze the effect of PPS as a potential adjunctive therapy for Fabry and Gaucher diseases.

Metabolic Labeling of Surface Neo-sialylglyconjugates Catalyzed by Trypanosoma cruzi trans-Sialidase.

Trypanosoma cruzi, the protozoan agent of Chagas disease, has evolved an innovative metabolic pathway by which protective sialic acid (SA) residues are scavenged from host sialylglycoconjugates and transferred onto parasite surface mucin-like molecules (or surface glycoconjugates from host target cells) by means of a unique trans-sialidase (TS) enzyme. TS-induced changes in the glycoprotein sialylation profile of both parasite and host cells are crucial for the establishment of a persistent T. cruzi infection and for the development of Chagas disease-associated pathogenesis. In this chapter, we describe a novel metabolic labeling method developed in our labs that enables straightforward identification and molecular characterization of SA acceptors of the TS-catalyzed reaction.

Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins.

BACKGROUND: TolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3-5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen. METHODS/PRINCIPLE FINDINGS: Genome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis. CONCLUSION/SIGNIFICANCE: This study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications.

Galectin-3 deficiency drives lupus-like disease by promoting spontaneous germinal centers formation via IFN-γ.

Germinal centers (GC) are important sites for high-affinity and long-lived antibody induction. Tight regulation of GC responses is critical for maintaining self-tolerance. Here, we show that Galectin-3 (Gal-3) is involved in GC development. Compared with WT mice, Gal-3 KO mice have more GC B cells and T follicular helper cells, increased percentages of antibody-secreting cells and higher concentrations of immunoglobulins and IFN-γ in serum, and develop a lupus-like disease. IFN-γ blockade in Gal-3 KO mice reduces spontaneous GC formation, class-switch recombination, autoantibody production and renal pathology, demonstrating that IFN-γ overproduction sustains autoimmunity. The results from chimeric mice show that intrinsic Gal-3 signaling in B cells controls spontaneous GC formation. Taken together, our data provide evidence that Gal-3 acts directly on B cells to regulate GC responses via IFN-γ and implicate the potential of Gal-3 as a therapeutic target in autoimmunity.

Next-generation ELISA diagnostic assay for Chagas Disease based on the combination of short peptidic epitopes.

Chagas Disease, caused by the protozoan Trypanosoma cruzi, is a major health and economic problem in Latin America for which no vaccine or appropriate drugs for large-scale public health interventions are yet available. Accurate diagnosis is essential for the early identification and follow up of vector-borne cases and to prevent transmission of the disease by way of blood transfusions and organ transplantation. Diagnosis is routinely performed using serological methods, some of which require the production of parasite lysates, parasite antigenic fractions or purified recombinant antigens. Although available serological tests give satisfactory results, the production of reliable reagents remains laborious and expensive. Short peptides spanning linear B-cell epitopes have proven ideal serodiagnostic reagents in a wide range of diseases. Recently, we have conducted a large-scale screening of T. cruzi linear B-cell epitopes using high-density peptide chips, leading to the identification of several hundred novel sequence signatures associated to chronic Chagas Disease. Here, we performed a serological assessment of 27 selected epitopes and of their use in a novel multipeptide-based diagnostic method. A combination of 7 of these peptides were finally evaluated in ELISA format against a panel of 199 sera samples (Chagas-positive and negative, including sera from Leishmaniasis-positive subjects). The multipeptide formulation displayed a high diagnostic performance, with a sensitivity of 96.3% and a specificity of 99.15%. Therefore, the use of synthetic peptides as diagnostic tools are an attractive alternative in Chagas' disease diagnosis.

The Trypomastigote Small Surface Antigen (TSSA) regulates Trypanosoma cruzi infectivity and differentiation.

BACKGROUND: TSSA (Trypomastigote Small Surface Antigen) is an antigenic, adhesion molecule displayed on the surface of Trypanosoma cruzi trypomastigotes. TSSA displays substantial sequence identity to members of the TcMUC gene family, which code for the trypomastigote mucins (tGPI-mucins). In addition, TSSA bears sequence polymorphisms among parasite strains; and two TSSA variants expressed as recombinant molecules (termed TSSA-CL and TSSA-Sy) were shown to exhibit contrasting features in their host cell binding and signaling properties. METHODS/PRINCIPLE FINDINGS: Here we used a variety of approaches to get insights into TSSA structure/function. We show that at variance with tGPI-mucins, which rely on their extensive O-glycoslylation to achieve their protective function, TSSA seems to be displayed on the trypomastigote coat as a hypo-glycosylated molecule. This has a functional correlate, as further deletion mapping experiments and cell binding assays indicated that exposition of at least two peptidic motifs is critical for the engagement of the 'adhesive' TSSA variant (TSSA-CL) with host cell surface receptor(s) prior to trypomastigote internalization. These motifs are not conserved in the 'non-adhesive' TSSA-Sy variant. We next developed transgenic lines over-expressing either TSSA variant in different parasite backgrounds. In strict accordance to recombinant protein binding data, trypomastigotes over-expressing TSSA-CL displayed improved adhesion and infectivity towards non-macrophagic cell lines as compared to those over-expressing TSSA-Sy or parental lines. These phenotypes could be specifically counteracted by exogenous addition of peptides spanning the TSSA-CL adhesion motifs. In addition, and irrespective of the TSSA variant, over-expression of this molecule leads to an enhanced trypomastigote-to-amastigote conversion, indicating a possible role of TSSA also in parasite differentiation. CONCLUSION/SIGNIFICANCE: In this study we provided novel evidence indicating that TSSA plays an important role not only on the infectivity and differentiation of T. cruzi trypomastigotes but also on the phenotypic variability displayed by parasite strains.

Osteocyte Alterations Induce Osteoclastogenesis in an In Vitro Model of Gaucher Disease.

Gaucher disease (GD) is caused by mutations in the glucosylceramidase ß (GBA 1) gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to the accumulation of the glycolipid glucocerebroside in the lysosomes of cells, mainly in the monocyte/macrophage lineage. Its mildest form is Type I GD, characterized by non-neuronopathic involvement. Bone compromise is the most disabling aspect of the Gaucher disease. However, the pathophysiological aspects of skeletal alterations are not yet fully understood. The bone tissue homeostasis is maintained by a balance between resorption of old bone by osteoclasts and new bone formation by osteoblasts. A central player in this balance is the osteocyte as it controls both processes. We studied the involvement of osteocytes in an in vitro chemical model of Gaucher disease. The osteocyte cell line MLO-Y4 was exposed to conduritol-ß-epoxide (CBE), an inhibitor of GCase, for a period of 7, 14 and 21 days. Conditioned media from CBE-treated osteocytes was found to induce osteoclast differentiation. GCase inhibition caused alterations in Cx43 expression and distribution pattern and an increase in osteocyte apoptosis. Osteoclast differentiation involved osteocyte apoptotic bodies, receptor activator of nuclear factor κ-B ligand (RANKL) and soluble factors. Thus, our results indicate that osteocytes may have a role to play in the bone pathophysiology of GD.

The Trypanosoma cruzi Surface, a Nanoscale Patchwork Quilt.

The Trypanosoma cruzi trypomastigote membrane provides a major protective role against mammalian host-derived defense mechanisms while allowing the parasite to interact with different cell types and trigger pathogenesis. This surface has been historically appreciated as a rather unstructured 'coat', mainly consisting of a continuous layer of glycolipids and heavily O-glycosylated mucins, occasionally intercalated with different developmentally regulated molecules displaying adhesive and/or enzymatic properties. Recent findings, however, indicate that the trypomastigote membrane is made up of multiple, densely packed and discrete 10-150nm lipid-driven domains bearing different protein composition; hence resembling a highly organized 'patchwork quilt' design. Here, we discuss different aspects underlying the biogenesis, assembly, and dynamics of this cutting-edge fashion outfit, as well as its functional implications.

Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.

Pathogenesis of Bone Alterations in Gaucher Disease: The Role of Immune System.

Gaucher, the most prevalent lysosomal disorder, is an autosomal recessive inherited disorder due to a deficiency of glucocerebrosidase. Glucocerebrosidase deficiency leads to the accumulation of glucosylceramide primarily in cells of mononuclear-macrophage lineage. Clinical alterations are visceral, hematological, and skeletal. Bone disorder in Gaucher disease produces defects on bone metabolism and structure and patients suffer from bone pain and crisis. Skeletal problems include osteopenia, osteoporosis, osteolytic lesions, and osteonecrosis. On the other hand a chronic stimulation of the immune system is a well-accepted hallmark in this disease. In this review we summarize the latest findings in the mechanisms leading to the bone pathology in Gaucher disease in relationship with the proinflammatory state.

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants.

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15 mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15 mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼ threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens.

Trypanosoma cruzi trans-sialidase prevents elicitation of Th1 cell response via interleukin 10 and downregulates Th1 effector cells.

The trans-sialidases (TSs) from Trypanosoma cruzi, the agent of Chagas disease, are virulence factors shed to the bloodstream that induce strong alterations in the immune system. Here, we report that both enzymatically active TS (aTS) and its lectinlike isoform (iTS) disturb CD4 T cell physiology, inducing downregulation of Th1 cell functionality and in vivo cell expansion. By using ovalbumin-specific DO11.10 cells as tracers of clones developing the Th1 phenotype, we found that the infection induced significant amounts of gamma interferon (IFN-γ) but low levels of interleukin 2 (IL-2) and increased IL-4 production in vivo, in agreement with a mixed T helper response. The production of cytokines associated with the Th2 phenotype was prevented by passive transfer of anti-TS neutralizing antibodies. TSs also reduced the T cell receptor signaling as assayed by Zap-70 phosphorylation. TSs also reduced IL-2 and IFN-γ secretion, with a concomitant increase in IL-4 production and then an unbalancing of the CD4 T cell response toward the Th2 phenotype. This effect was prevented by using anti-IL-10 neutralizing antibodies or IL-10(-/-) antigen-presenting cells, supporting the subversion of this regulatory pathway. In support, TSs stimulated IL-10 secretion by antigen-presenting cells during their interaction with CD4 T cells. When polarized cells were stimulated in the presence of TSs, the secretion of IL-2 and IFN-γ was strongly downregulated in Th1 cells, while IL-2 production was upregulated in Th2 cells. Although the Th1 response is associated with host survival, it may simultaneously induce extensive damage to infected tissues. Thus, by delaying the elicitation of the Th1 response and limiting its effector properties, TSs restrain the cell response, supporting T. cruzi colonization and persistence while favoring host survival.

Rab11 regulates trafficking of trans-sialidase to the plasma membrane through the contractile vacuole complex of Trypanosoma cruzi.

Trypanosoma cruzi is the etiologic agent of Chagas disease. Although this is not a free-living organism it has conserved a contractile vacuole complex (CVC) to regulate its osmolarity. This obligate intracellular pathogen is, in addition, dependent on surface proteins to invade its hosts. Here we used a combination of genetic and biochemical approaches to delineate the contribution of the CVC to the traffic of glycosylphosphatidylinositol (GPI)-anchored proteins to the plasma membrane of the parasite and promote host invasion. While T. cruzi Rab11 (GFP-TcRab11) localized to the CVC, a dominant negative (DN) mutant tagged with GFP (GFP-TcRab11DN) localized to the cytosol, and epimastigotes expressing this mutant were less responsive to hyposmotic and hyperosmotic stress. Mutant parasites were still able to differentiate into metacyclic forms and infect host cells. GPI-anchored trans-sialidase (TcTS), mucins of the 60-200 KDa family, and trypomastigote small surface antigen (TcTSSA II) co-localized with GFP-TcRab11 to the CVC during transformation of intracellular amastigotes into trypomastigotes. Mucins of the gp35/50 family also co-localized with the CVC during metacyclogenesis. Parasites expressing GFP-TcRab11DN prevented TcTS, but not other membrane proteins, from reaching the plasma membrane, and were less infective as compared to wild type cells. Incubation of these mutants in the presence of exogenous recombinant active, but not inactive, TcTS, and a sialic acid donor, before infecting host cells, partially rescued infectivity of trypomastigotes. Taking together these results reveal roles of TcRab11 in osmoregulation and trafficking of trans-sialidase to the plasma membrane, the role of trans-sialidase in promoting infection, and a novel unconventional mechanism of GPI-anchored protein secretion.

Uncoupling of osteoblast-osteoclast regulation in a chemical murine model of Gaucher disease.

Gaucher disease (GD) is caused by mutations in the GBA gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to accumulation of the glycolipid glucocerebroside in the lysosomes of cells of monocyte/macrophage system. Type I GD is the mildest form and is characterized by the absence of neuronopathic affection. Bone compromise in Gaucher disease patients is the most disabling aspect of the disease. However, pathophysiological aspects of skeletal alterations are still poorly understood. The homeostasis of bone tissue is maintained by the balanced processes of bone resorption by osteoclasts and formation by osteoblasts. We decided to test whether bone resorption and/or bone formation could be altered by the use of a chemical in vitro murine model of Gaucher disease. We used two sources of cells from monocyte/macrophages lineage isolated from normal mice, splenocytes (S) and peritoneal macrophages (PM), and were exposed to CBE, the inhibitor of GCase (S-CBE and PM-CBE, respectively). Addition of both conditioned media (CM) from S-CBE and PM-CBE induced the differentiation of osteoclasts precursors from bone marrow to mature and functional osteoclasts. TNF-α could be one of the factors responsible for this effect. On the other hand, addition of CM to an osteoblast cell culture resulted in a reduction in expression of alkaline phosphatase and mineralization process. In conclusion, these results suggest implication of changes in both bone formation and bone resorption and are consistent with the idea that both sides of the homeostatic balance are affected in GD.

Trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors RORγt and Ahr that leads to IL-17 production by activated B cells.

Here we identified B cells as a major source of rapid, innate-like production of interleukin 17 (IL-17) in vivo in response to infection with Trypanosoma cruzi. IL-17(+) B cells had a plasmablast phenotype, outnumbered cells of the TH17 subset of helper T cells and were required for an optimal response to this pathogen. With both mouse and human primary B cells, we found that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell-surface mucin CD45, which led to signaling dependent on the kinase Btk and production of IL-17A or IL-17F via a transcriptional program independent of the transcription factors RORγt and Ahr. Our combined data suggest that the generation of IL-17(+) B cells may be a previously unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.