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All healthy humans have high levels of natural anti-α-galactosyl (α-Gal) antibodies (elicited by yet uncharacterized glycotopes), which may play important roles in immunoglycomics: (a) potential protection against certain parasitic and viral zoonotic infections; (b) targeting of α-Gal-engineered cancer cells; (c) aiding in tissue repair; and (d) serving as adjuvants in α-Gal-based vaccines. Patients with certain protozoan infections have specific anti-α-Gal antibodies, elicited against parasite-derived α-Gal-bearing glycotopes. These glycotopes, however, remain elusive except for the well-characterized glycotope Galα1,3Galβ1,4GlcNAcα, expressed by Trypanosoma cruzi. The discovery of new parasitic glycotopes is greatly hindered by the enormous structural diversity of cell-surface glycans and the technical challenges of classical immunoglycomics, a top-down approach from cultivated parasites to isolated glycans. Here, we demonstrate that reversed immunoglycomics, a bottom-up approach, can identify parasite species-specific α-Gal-bearing glycotopes by probing synthetic oligosaccharides on neoglycoproteins. This method was tested here seeking to identify as-yet unknown glycotopes specific for Leishmania major, the causative agent of Old-World cutaneous leishmaniasis (OWCL). Neoglycoproteins decorated with synthetic α-Gal-containing oligosaccharides derived from L. major glycoinositolphospholipids served as antigens in a chemiluminescent enzyme-linked immunosorbent assay using sera from OWCL patients and noninfected individuals. Receiver-operating characteristic analysis identified Galpα1,3Galfβ and Galpα1,3Galfβ1,3Manpα glycotopes as diagnostic biomarkers for L. major-caused OWCL, which can distinguish with 100% specificity from heterologous diseases and L. tropica-caused OWCL. These glycotopes could prove useful in the development of rapid α-Gal-based diagnostics and vaccines for OWCL. Furthermore, this method could help unravel cryptic α-Gal-glycotopes of other protozoan parasites and enterobacteria that elicit the natural human anti-α-Gal antibodies.

Chronic Chagas cardiomyopathy (CCC) is the most frequent and severe form of Chagas disease, a neglected tropical illness caused by the protozoan Trypanosoma cruzi, and the main cause of morbimortality from cardiovascular problems in endemic areas. Although efforts have been made to understand the signaling pathways and molecular mechanisms underlying CCC, the immunological signaling pathways regulated by the etiological treatment with benznidazole (Bz) has not been reported. In experimental CCC, Bz combined with the hemorheological and immunoregulatory agent pentoxifylline (PTX) has beneficial effects on CCC. To explore the molecular mechanisms of Bz or Bz+PTX therapeutic strategies, C57BL/6 mice chronically infected with the T. cruzi Colombian strain (discrete typing unit TcI) and showing electrocardiographic abnormalities were submitted to suboptimal dose of Bz or Bz+PTX from 120 to 150 days postinfection. Electrocardiographic alterations, such as prolonged corrected QT interval and heart parasite load, were beneficially impacted by Bz and Bz+PTX. RT-qPCR TaqMan array was used to evaluate the expression of 92 genes related to the immune response in RNA extracted from heart tissues. In comparison with non-infected mice, 30 genes were upregulated, and 31 were downregulated in infected mice. Particularly, infection upregulated the cytokines IFN-γ, IL-12b, and IL-2 (126-, 44-, and 18-fold change, respectively) and the T-cell chemoattractants CCL3 and CCL5 (23- and 16-fold change, respectively). Bz therapy restored the expression of genes related to inflammatory response, cellular development, growth, and proliferation, and tissue development pathways, most probably linked to the cardiac remodeling processes inherent to CCC, thus mitigating the Th1-driven response found in vehicle-treated infected mice. The combined Bz+PTX therapy revealed pathways related to the modulation of cell death and survival, and organismal survival, supporting that this strategy may mitigate the progression of CCC. Altogether, our results contribute to the better understanding of the molecular mechanisms of the immune response in the heart tissue in chronic Chagas disease and reinforce that parasite persistence and dysregulated immune response underpin CCC severity. Therefore, Bz and Bz+PTX chemotherapies emerge as tools to interfere in these pathways aiming to improve CCC prognosis.

descripción no proporcionada por scopus, ISGlobal work is supported by the Departament d’Universitats i Recerca de la Generalitat de Catalunya, Spain (AGAUR; 017SGR00924) and by the Instituto de Salud Carlos III (ISCIII) RICET Network for Cooperative Research in Tropical Diseases (ISCIII; RD16/0027/0004 - PI1290) and FEDER. MJP research is supported by the Ministry of Health, Government of Catalonia (PERIS 2016-2010 SLT008/18/00132). ICA, JG, and FT are supported by the grant number U01AI129783 from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). ICA is also partly supported by the grant number 5U54MD007592 from the National Institute on Minority Health and Health Disparities (NIMHD), NIH. MCL and MCT were supported by ISCIII RICET grant RD16/0027/0005 - PI1290 and FEDER and by grants SAF2016-81003-R and SAF2016-80998-R from the Spanish “Programa Estatal I+D+i (MINECO)”. AA's work was supported by the Italian Ministry of Health “Fondi Ricerca Corrente - Linea 3, progetto 9” to IRCCS Sacro Cuore Don Calabria Hospital. JR was supported by CONACyT Fossis grant #261006. The Drugs for Neglected Diseases initiative (DNDi) is grateful to its donors, public and private, who have provided funding to DNDi since its inception in 2003. A full list of DNDi's donors can be found at http://www.dndi.org/donate/donors/. FIND is grateful to its donors, public and private, who have helped bring innovative new diagnostics for diseases of poverty. A full list of FIND’s donors can be found at: https://www.finddx.org/partners-donors/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The microneme organelles of Toxoplasma gondii tachyzoites release protein complexes (MICs), including one composed of the transmembrane protein MIC6 plus MIC1 and MIC4. In this complex, carbohydrate recognition domains of MIC1 and MIC4 are exposed and interact with terminal sialic acid and galactose residues, respectively, of host cell glycans. Recently, we demonstrated that MIC1 and MIC4 binding to the N-glycans of Toll-like receptor (TLR) 2 and TLR4 on phagocytes triggers cell activation and pro-inflammatory cytokine production. Herein, we investigated the requirement for TLR2 heterodimerization and co-receptors in MIC-induced responses, as well as the signaling molecules involved. We used MICs to stimulate macrophages and HEK293T cells transfected with TLR2 and TLR1 or TLR6, both with or without the co-receptors CD14 and CD36. Then, the cell responses were analyzed, including nuclear factor-kappa B (NF-&kappa, B) activation and cytokine production, which showed that (1) only TLR2, among the studied factors, is crucial for MIC-induced cell activation, (2) TLR2 heterodimerization augments, but is not critical for, activation, (3) CD14 and CD36 enhance the response to MIC stimulus, and (4) MICs activate cells through a transforming growth factor beta-activated kinase 1 (TAK1)-, mammalian p38 mitogen-activated protein kinase (p38)-, and NF-&kappa, B-dependent pathway. Remarkably, among the studied factors, the interaction of MIC1 and MIC4 with TLR2 N-glycans is sufficient to induce cell activation, which promotes host protection against T. gondii infection.

Trypanosoma bruceispp. develop into mammalian-infectious metacyclic trypomastigotes inside the tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, nothing is known about expression of invariant surface antigens by the metacyclic stage. Proteomic analysis of saliva fromT. brucei-infected flies revealed a novel family of hypothetical GPI-anchored surface proteins herein named Metacyclic Invariant Surface Proteins (MISP). MISP are encoded by five homolog genes and share ~80% protein identity. The crystal structure of MISP N-terminus at 1.82 Å resolution revealed a triple helical bundle that shares key features with other trypanosome surface proteins. However, molecular modelling combined with live fluorescent microscopy suggest that MISP N-termini are extended above the metacyclic VSG coat, exposing immunogenic epitopes. Collectively, we suggest that the metacyclic cell surface architecture appears more permissive than bloodstream forms in terms of expression of invariant GPI-anchored glycoproteins, which could be exploited for the development of novel vaccines against African trypanosomiases.

Parasitic diseases affect billions of people and are considered a major public health issue. Close to 400 species are estimated to parasitize humans, of which around 90 are responsible for great clinical burden and mortality rates. Unfortunately, they are largely neglected as they are mainly endemic to poor regions. Of relevance to this review, there is accumulating evidence of the release of extracellular vesicles (EVs) in parasitic diseases, acting both in parasite parasite inter-communication as well as in parasite host interactions. EVs participate in the dissemination of the pathogen and play a role in the regulation of the host immune systems. Production of EVs from parasites or parasitized cells has been described for a number of parasitic infections. In this review, we provide the most relevant findings of the involvement of EVs in intercellular communication, modulation of immune responses, involvement in pathology, and their potential as new diagnostic tools and therapeutic agents in some of the major human parasitic pathogens., National Council for Scientific and Technological Development (CNPq), United States Department of Health & Human Services National Institutes of Health (NIH) - USA R01AI070655-A5 R01AI070655-A5S1 2G12MD007592, European Union (EU), Ministerio Español de Economía y Competitividad

Background Protozoan parasites from the genus Leishmania cause broad clinical manifestations known as leishmaniases, which affect millions of people worldwide. Cutaneous leishmaniasis (CL), caused by L. major, is one the most common forms of the disease in the Old World. There is no preventive or therapeutic human vaccine available for L. major CL, and existing drug treatments are expensive, have toxic side effects, and resistant parasite strains have been reported. Hence, further therapeutic interventions against the disease are necessary. Terminal, non-reducing, and linear α-galactopyranosyl (α-Gal) epitopes are abundantly found on the plasma membrane glycolipids of L. major known as glycoinositolphospholipids. The absence of these α-Gal epitopes in human cells makes these glycans highly immunogenic and thus potential targets for vaccine development against CL. Methodology/Principal findings Here, we evaluated three neoglycoproteins (NGPs), containing synthetic α-Gal epitopes covalently attached to bovine serum albumin (BSA), as vaccine candidates against L. major, using α1,3-galactosyltransferase-knockout (α1,3GalT-KO) mice. These transgenic mice, similarly to humans, do not express nonreducing, linear α-Gal epitopes in their cells and are, therefore, capable of producing high levels of anti-α-Gal antibodies. We observed that Galα(1,6)Galβ-BSA (NGP5B), but not Galα(1,4)Galβ-BSA (NGP12B) or Galα(1,3)Galα-BSA (NGP17B), was able to significantly reduce the size of footpad lesions by 96% in comparison to control groups. Furthermore, we observed a robust humoral and cellular immune response with production of high levels of protective lytic anti-α-Gal antibodies and induction of Th1 cytokines. Conclusions/Significance We propose that NGP5B is an attractive candidate for the study of potential synthetic α-Gal-neoglycoprotein-based vaccines against L. major infection., Author summary Despite a worldwide prevalence, cutaneous leishmaniasis (CL) remains largely neglected, with no prophylactic or therapeutic vaccine available. In the Old World, CL is mainly caused by either Leishmania major or L. tropica parasites, which produce localized cutaneous ulcers, often leading to scarring and social stigma. Currently, the disease has reached hyperendemicity levels in the Middle East due to conflict and human displacement. Furthermore, the first choice of treatment in that region continues to be pentavalent antimonials, which are costly and highly toxic, and current vector control measures alone are not sufficient to stop disease transmission. Hence, a vaccine against CL would be very beneficial. Previous studies have demonstrated that sugars are promising vaccine candidates against leishmaniasis, since most parasite species have a cell surface coat composed of immunogenic sugars, including linear α-galactopyranosyl (α-Gal) epitopes, which are absent in humans. Here, we have developed an α-Gal-based vaccine candidate, named NGP5B. When tested in transgenic mice which like humans lack α-Gal epitopes in their cells, NGP5B was able to induce a significant partial protection against L. major infection, by significantly reducing mouse footpad lesions and parasite burden. Altogether, we propose NGP5B as a promising preventive vaccine for CL caused by L. major.

Cilia play important roles in cell signaling, facilitated by the unique lipid environment of a ciliary membrane containing high concentrations of sterol-rich lipid rafts. The African trypanosome Trypanosoma brucei is a single-celled eukaryote with a single cilium/flagellum. We tested whether flagellar sterol enrichment results from selective flagellar partitioning of specific sterol species or from general enrichment of all sterols. While all sterols are enriched in the flagellum, cholesterol is especially enriched. T. brucei cycles between its mammalian host (bloodstream cell), in which it scavenges cholesterol, and its tsetse fly host (procyclic cell), in which it both scavenges cholesterol and synthesizes ergosterol. We wondered whether the insect and mammalian life cycle stages possess chemically different lipid rafts due to different sterol utilization. Treatment of bloodstream parasites with cholesterol-specific methyl-β-cyclodextrin disrupts both membrane liquid order and localization of a raft-associated ciliary membrane calcium sensor. Treatment with ergosterol-specific amphotericin B does not. The opposite results were observed with ergosterol-rich procyclic cells. Further, these agents have opposite effects on flagellar sterol enrichment and cell metabolism in the two life cycle stages. These findings illuminate differences in the lipid rafts of an organism employing life cycle-specific sterols and have implications for treatment.

Made available in DSpace on 2018-11-26T16:16:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-08-10 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) FAPERGS (Fundacao de Amparo a Pesquisa do Estado do Rio Grande do Sul) PRONEX (Programa de Nucleos de Excelencia) In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a wellknown inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new molecular mechanism of resistance to pesticides. Inst Oswaldo Cruz, Lab Microbiol Celular, BR-20001 Rio De Janeiro, Brazil Univ Fed Rio Grande do Sul, Ctr Biotecnol, Porto Alegre, RS, Brazil Univ Fed Rio Grande do Sul, Fac Vet, Porto Alegre, RS, Brazil Univ Fed Rio de Janeiro, Inst Bioquim Med Leopoldo Meis, Programa Biol Mol Biotecnol, BR-20001 Rio de Janeiro, Brazil Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Patol Vet, Jaboticabal, Brazil INCTEM, Rio De Janeiro, Brazil Univ Texas El Paso, Border Biomed Res Ctr, El Paso, TX 79968 USA Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Patol Vet, Jaboticabal, Brazil

The definition of a biomarker provided by the World Health Organization is any substance, structure, or process that can be measured in the body, or its products and influence, or predict the incidence or outcome of disease. Currently, the lack of prognosis and progression markers for chronic Chagas disease has posed limitations for testing new drugs to treat this neglected disease. Several molecules and techniques to detect biomarkers in Trypanosoma cruzi-infected patients have been proposed to assess whether specific treatment with benznidazole or nifurtimox is effective. Isolated proteins or protein groups from different T. cruzi stages and parasite-derived glycoproteins and synthetic neoglycoconjugates have been demonstrated to be useful for this purpose, as have nucleic acid amplification techniques. The amplification of T. cruzi DNA using the real-time polymerase chain reaction method is the leading test for assessing responses to treatment in a short period of time. Biochemical biomarkers have been tested early after specific treatment. Cytokines and surface markers represent promising molecules for the characterisation of host cellular responses, but need to be further assessed., RICET RD12/0018/0010. RICET RD12/0018/0021. AGAUR 2014SGR26. Plan Nacional de I+D+I SAF2012-35777. Plan Nacional de I+D+I SAF2013-48527-R. NIMHD/NIH 2G12MD007592. Financial support: CRESIB and IPBLN research members were partially supported by the RICET (RD12/0018/0010, RD12/0018/0021), M-JP and JG received research funds from AGAUR (2014SGR26) and Fundación Mundo Sano, M-CT and M-CL were supported by Plan Nacional de I+D+I (MINECO-Spain) (SAF2012-35777, SAF2013-48527-R and FEDER), ICA was partially supported by NIMHD/NIH (2G12MD007592). Financial support: CRESIB and IPBLN research members were partially supported by the RICET (RD12/0018/0010, RD12/0018/0021), M-JP and JG received research funds from AGAUR (2014SGR26) and Fundación Mundo Sano, M-CT and M-CL were supported by Plan Nacional de I+D+I (MINECO-Spain) (SAF2012-35777, SAF2013-48527-R and FEDER), ICA was partially supported by NIMHD/NIH (2G12MD007592).

Anthrax toxin receptors act as molecular clamps or switches that control anthrax toxin entry, pH-dependent pore formation, and translocation of enzymatic moieties across the endosomal membranes. We previously reported that reduction of the disulfide bonds in the immunoglobulin-like (Ig) domain of the anthrax toxin receptor 2 (ANTXR2) inhibited the function of the protective antigen (PA) pore. In the present study, the disulfide linkage in the Ig domain was identified as Cys255-Cys279 and Cys230-Cys315. Specific disulfide bond deletion mutants were achieved by replacing Cys residues with Ala residues. Deletion of the disulfide bond C255-C279, but not C230-C315, inhibited the PA pore-induced release of the fluorescence dyes from the liposomes, suggesting that C255-C279 is essential for PA pore function. Furthermore, we found that deletion of C255-C279 did not affect PA prepore-to-pore conversion, but inhibited PA pore membrane insertion by trapping the PA membrane-inserting loops in proteinaceous hydrophobic pockets. Fluorescence spectra of Trp59, a residue adjacent to the PA-binding motif in von Willebrand factor A (VWA) domain of ANTXR2, showed that deletion of C255-C279 resulted in a significant conformational change on the receptor ectodomain. The disulfide deletion-induced conformational change on the VWA domain was further confirmed by single-particle 3D reconstruction of the negatively stained PA-receptor heptameric complexes. Together, the biochemical and structural data obtained in this study provides a mechanistic insight into the role of the receptor disulfide bond C255-C279 in anthrax toxin action. Manipulation of the redox states of the receptor, specifically targeting to C255-C279, may become a novel strategy to treat anthrax.

TLR2 plays a critical role in the protection against Paracoccidioides brasiliensis conferred by ArtinM administration. ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus, induces IL-12 production in macrophages and dendritic cells, which accounts for the T helper1 immunity that results from ArtinM administration. We examined the direct interaction of ArtinM with TLR2using HEK293A cells transfected with TLR2, alone or in combination with TLR1 or TLR6, together with accessory proteins. Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6. Murine macrophages that were stimulated with ArtinM had augmented TLR2 mRNA expression. Furthermore, pre-incubation of unstimulated macrophages with an anti-TLR2 antibody reduced the cell labeling with ArtinM. In addition, a microplate assay revealed that ArtinM bound to TLR2 molecules that had been captured by specific antibodies from a macrophages lysate. Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose. The biological relevance of the direct interaction of ArtinM with TLR2 glycans was assessed using macrophages from TLR2-KOmice, which produced significantly lower levels of IL-12 and IL-10 in response to ArtinM than macrophages from wild-type mice. Pre-treatment of murine macrophages with pharmacological inhibitors of signaling molecules demonstrated the involvement of p38 MAPK and JNK in the IL-12 production induced by ArtinM and the involvement ofPI3K in IL-10 production. Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties.

Departamento de Parasitologia, ICB2, USP, 05508-900 Sao Paulo, SP, Brasil *Departamento de Bioquimica eImmunologia, UFMG, Belo Horizonte, MG, Brasil **Laboratorio de Doenca de Chagas, CPqRR-Fiocruz,Belo Horizonte, MG, Brasil ***Department of Biochemistry, University of Dundee, Dundee, Scotland,UnitedKingdom ****Departamento de Immunologia, Microbiologia e Parasitologia, Unifesp, Sao Paulo, SP, BrasilKey words: Trypanosoma cruzi - mucins - Chagas disease

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) NIH Background: the fungal cell wall is a complex and dynamic outer structure. in pathogenic fungi its components interact with the host, determining the infection fate. the present work aimed to characterize cell wall lipids from P. brasiliensis grown in the presence and absence of human plasma. We compared the results from isolates Pb3 and Pb18, which represent different phylogenetic species that evoke distinct patterns of experimental paracoccidioidomycosis.Methodology/Principal Findings: We comparatively characterized cell wall phospholipids, fatty acids, sterols, and neutral glycolipids by using both electrospray ionization- and gas chromatography-mass spectrometry analyses of lipids extracted with organic solvents followed by fractionation in silica-gel-60. We detected 49 phospholipid species in Pb3 and 38 in Pb18, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. in both Pb3 and Pb18, PC and PE had the most numerous species. Among the fatty acids, C18:1 and C18:2 were the most abundant species in both isolates, although C18:2 was more abundant in Pb18. There was a different effect of plasma supplementation on fatty acids depending on the fungal isolate. the prevalent glycolipid species was HexC18:0-OH/d19:2-Cer, although other four minor species were also detected. the most abundant sterol in all samples was brassicasterol. Distinct profiles of cell wall and total yeast sterols suggested that the preparations were enriched for cell wall components. the presence of plasma in the culture medium specially increased cell wall brassicasterol abundance and also other lipids.Conclusions/Significance: We here report an original comparative lipidomic analysis of P. brasiliensis cell wall. Our results open doors to understanding the role of cell wall lipids in fungal biology, and interaction with anti-fungal drugs and the host. Universidade Federal de São Paulo, UNIFESP, EPM, Dept Microbiol Imunol & Parasitol, São Paulo, Brazil Univ Texas El Paso, Dept Biol Sci, Border Biomed Res Ctr, El Paso, TX 79968 USA Universidade Federal de São Paulo, UNIFESP, EPM, Dept Microbiol Imunol & Parasitol, São Paulo, Brazil NIH: G12MD007592 NIH: 5G12RR008124-16A1 NIH: 5G12RR008124-16A1S1 Web of Science

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) National Institutes of Health Biomolecule Analysis Core Facility at the Border Biomedical Research Center/Biology/UTEP University of Texas at El Paso (National Institutes of Health) The glycosylphosphatidylinositol (GPI)-anchored mucins of Trypanosoma cruzi trypomastigotes play an important immunomodulatory role during the course of Chagas disease. Here, some biological activities of tGPI-mucins from four T cruzi isolates, including benznidazole-susceptible (BZS-Y), benznidazole-resistant (BZR-Y), CL, and Colombiana, were evaluated. GPI-mucins were able to differentially trigger the production of interleukin-12 and nitric oxide in BALB/c macrophages and modulate LLC-MK2 cell invasion. the significance of these variations was assessed after analysis of the terminal alpha-galactosyl residues. Enzymatic treatment with alpha-galactosidase indicated a differential expression of O-linked alpha-galactosyl residues among the strains, with higher expression of this sugar in BZS-Y and BZR-Y T cruzi populations followed by Colombiana and CL. Unweighted pair group method analysis of the carbohydrate anchor profile and biological parameters allowed the clustering of two groups. One group includes Y and CL strains (T cruzi II and VI), and the other group is represented by Colombiana strain (T. cruzi I). Fiocruz MS, Ctr Pesquisas Rene Rachou, BR-30190002 Belo Horizonte, MG, Brazil Universidade Federal de São Paulo, UNIFESP, Dept Ciencias Biol, Diadema, SP, Brazil Univ Texas El Paso, Dept Biol Sci, Border Biomed Res Ctr, El Paso, TX 79968 USA Universidade Federal de São Paulo, UNIFESP, Dept Ciencias Biol, Diadema, SP, Brazil CNPq: 305042/2010-6 CNPq: 552072/2009-5 CNPq: PAPES-IV-400138/2006-9 CNPq: PDJ-150880/2005-7 National Institutes of Health: 1R01AI070655-04 National Institutes of Health: 3R01AI070655-04S1 National Institutes of Health: 2G12RR008124-16A1 National Institutes of Health: 2G12RR008124-16A1S1 Biomolecule Analysis Core Facility at the Border Biomedical Research Center/Biology/UTEP University of Texas at El Paso (National Institutes of Health): 2G12RR008124-16A1 Biomolecule Analysis Core Facility at the Border Biomedical Research Center/Biology/UTEP University of Texas at El Paso (National Institutes of Health): 2G12RR008124-16A1S1 Biomolecule Analysis Core Facility at the Border Biomedical Research Center/Biology/UTEP University of Texas at El Paso (National Institutes of Health): G12MD007592 Web of Science

SUMOylation is a relevant protein post-translational modification in eukaryotes. The C terminus of proteolytically activated small ubiquitin-like modifier (SUMO) is covalently linked to a lysine residue of the target protein by an isopeptide bond, through a mechanism that includes an E1-activating enzyme, an E2-conjugating enzyme, and transfer to the target, sometimes with the assistance of a ligase. The modification is reversed by a protease, also responsible for SUMO maturation. A number of proteins have been identified as SUMO targets, participating in the regulation of cell cycle progression, transcription, translation, ubiquitination, and DNA repair. In this study, we report that orthologous genes corresponding to the SUMOylation pathway are present in the etiological agent of Chagas disease, Trypanosoma cruzi. Furthermore, the SUMOylation system is functionally active in this protozoan parasite, having the requirements for SUMO maturation and conjugation. Immunofluorescence analysis showed that T. cruzi SUMO (TcSUMO) is predominantly found in the nucleus. To identify SUMOylation targets and get an insight into their physiological roles we generated transfectant T. cruzi epimastigote lines expressing a double- tagged T. cruzi SUMO, and SUMOylated proteins were enriched by tandem affinity chromatography. By two-dimensional liquid chromatography-mass spectrometry a total of 236 proteins with diverse biological functions were identified as potential T. cruzi SUMO targets. Of these, metacaspase-3 was biochemically validated as a bona fide SUMOylation substrate. Proteomic studies in other organisms have reported that orthologs of putative T. cruzi SUMOylated proteins are similarly modified, indicating conserved functions for protein SUMOylation in this early divergent eukaryote. Fil: Bayona, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Nakayasu, Ernesto S.. University of Texas at El Paso; Estados Unidos Fil: Laverriere, Marc. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Aguilar, Clemente. University of Texas at El Paso; Estados Unidos Fil: Sobreira, Tiago J. P.. Universidade de Sao Paulo; Brasil Fil: Choi, Hyungwon. University of Michigan; Estados Unidos Fil: Nesvizhskii, Alexey I.. University of Michigan; Estados Unidos Fil: Almeida, Igor C.. University of Texas at El Paso; Estados Unidos Fil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Alvarez, Vanina Eder. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Korean Government (MEST) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) NIH The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. the screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. the 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. in addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy. Inst Pasteur Korea, Image Min Grp, Songnam, Gyeonggi Do, South Korea Inst Pasteur Korea, Ctr Neglected Dis Drug Discovery, Songnam, Gyeonggi Do, South Korea Inst Pasteur Korea, Discovery Biol Grp, Songnam, Gyeonggi Do, South Korea Yonsei Univ, Dept Biochem, Seoul 120749, South Korea Yonsei Univ, Natl Res Lab, Seoul 120749, South Korea Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, Brazil Univ Texas El Paso, Dept Biol Sci, Border Biomed Res Ctr, El Paso, TX 79968 USA CSIR Biosci, Synthet Biol ERA, High Throughput Biol Grp, Pretoria, South Africa Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, Brazil Korean Government (MEST): K204EA000001-09E0100-00110 NIH: 2S06GM00812 NIH: 1R01AI070655 NIH: 5G12RR008124-16A1 Web of Science

Exosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such as Cryptococcus neoformans and Histoplasma capsulatum . Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes in Paracoccidioides brasiliensis. P. brasiliensis is a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 × g. P. brasiliensis antigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, with Marasmius oreades agglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. In P. brasiliensis cells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role played in vivo by vesicle components and α-galactosyl epitopes.

Heat shock proteins (Hsps) are among the most widely distributed and evolutionary conserved proteins. Hsps are essential regulators of diverse constitutive metabolic processes and are markedly upregulated during stress. A 62 kDa Hsp (Hsp60) of Histoplasma capsulatum (Hc) is an immunodominant antigen and the major surface ligand to CR3 receptors on macrophages. However little is known about the function of this protein within the fungus. We characterized Hc Hsp60-protein interactions under different temperature to gain insights of its additional functions oncell wall dynamism, heat stress and pathogenesis. We conducted co-immunoprecipitations with antibodies to Hc Hsp60 using cytoplasmic and cell wall extracts. Interacting proteins were identified by shotgun proteomics. For the cell wall, 84 common interactions were identified among the 3 growth conditions, including proteins involved in heat-shock response, sugar and amino acid/protein metabolism and cell signaling. Unique interactions were found at each temperature [30°C (81 proteins), 37°C (14) and 37/40°C (47)]. There were fewer unique interactions in cytoplasm [30°C (6), 37°C (25) and 37/40°C (39)] and four common interactions, including additional Hsps and other known virulence factors. These results show the complexity of Hsp60 function and provide insights into Hc biology, which may lead to new avenues for the management of histoplasmosis.

Background Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'. Conclusions BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

The cellular events required for unconventional protein secretion in eukaryotic pathogens are beginning to be revealed. In fungi, extracellular release of proteins involves passage through the cell wall by mechanisms that are poorly understood. In recent years, several studies demonstrated that yeast cells produce vesicles that traverse the cell wall to release a wide range of cellular components into the extracellular space. These studies suggested that extracellular vesicle release involves components of both conventional and unconventional secretory pathways, although the precise mechanisms required for this process are still unknown. We discuss here cellular events that are candidates for regulating this interesting but elusive event in the biology of yeast cells.

Reservosomes are the endpoint of the endocytic pathway in Trypanosoma cruzi epimastigotes. These organelles have the particular ability to concentrate proteins and lipids obtained from medium together with the main proteolytic enzymes originated from the secretory pathway, being at the same time a storage organelle and the main site of protein degradation. Subcellular proteomics have been extensively used for profiling organelles in different cell types. Here, we combine cell fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify reservosome-resident proteins. Starting from a purified reservosome fraction, we established a protocol to isolate reservosome membranes. Transmission electron microscopy was applied to confirm the purity of the fractions. To achieve a better coverage of identified proteins we analyzed the fractions separately and combined the results. LC-MS/MS analysis identified in total 709 T. cruzi-specific proteins; of these, 456 had predicted function and 253 were classified as hypothetical proteins. We could confirm the presence of most of the proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins and others. The definition of the reservosome protein profile is a good tool to assess their molecular signature, identify molecular markers, and understand their relationship with different organelles.

The pathogenic protozoan Giardia lamblia is known to not synthesize membrane lipids de novo. Therefore, it is possible that lipids in the small intestine, where trophozoites colonize, play key roles in regulating the growth and differentiation of this important pathogen. The focus of the current study is to conduct a complete lipidomic analysis and to test the hypothesis that Giardia has some ability to generate new phospholipids (PLs). Using mass spectrometry, now we show that phosphatidylglycerols (PGs) are major PLs followed by phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in non-encysting and encysting trophozoites, as well in cysts. The fatty acids attached to these PLs consist mostly of palmitate, palmitoleate, oleate, and linoleate. Results also indicate that PGs and PEs, unlike PCs, are not present in bovine bile and serum, the major sources of lipids of the culture medium, and that they could therefore be produced by fatty acid and headgroup remodeling reactions, circumventing the synthesis of entirely new PLs via de novo pathways. Genomic and transcriptional analyses show the presence of giardial phosphatidylglycerolphosphate synthase (gpgs) and phosphatidylserine decarboxylase (gpsd) genes, which are expressed throughout the life cycle. Bioinformatic and phylogenetic analyses further indicated that both genes are of prokaryotic origin and that they have undergone duplication in the course of the evolution. Our studies suggest that the abundance of PG in Giardia is unique among eukaryotes and that its synthesis thus could serve as a potential target for developing new therapies against this waterborne parasite.

Cryptococcus neoformans produces vesicles containing its major virulence factor, the capsular polysaccharide glucuronoxylomannan (GXM). These vesicles cross the cell wall to reach the extracellular space, where the polysaccharide is supposedly used for capsule growth or delivered into host tissues. In the present study, we characterized vesicle morphology and protein composition by a combination of techniques including electron microscopy, proteomics, enzymatic activity, and serological reactivity. Secretory vesicles in C. neoformans appear to be correlated with exosome-like compartments derived from multivesicular bodies. Extracellular vesicles manifested various sizes and morphologies, including electron-lucid membrane bodies and electron-dense vesicles. Seventy-six proteins were identified by proteomic analysis, including several related to virulence and protection against oxidative stress. Biochemical tests indicated laccase and urease activities in vesicles. In addition, different vesicle proteins were recognized by sera from patients with cryptococcosis. These results reveal an efficient and general mechanism of secretion of pathogenesis-related molecules in C. neoformans , suggesting that extracellular vesicles function as “virulence bags” that deliver a concentrated payload of fungal products to host effector cells and tissues.

BackgroundAll organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein fromstarved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted fromSalmonella entericaserovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column.ResultsTheN-terminal sequencing of the jacalin-bound protein revealed 100% identity with the Dps ofS. entericaserovar Typhimurium. Methyl-alpha-galactopyranoside inhibited the binding of Dps to jacalin in an enzyme-linked lectin assay, suggesting that the carbohydrate recognition domain (CRD) of jacalin is involved in the interaction with Dps. Furthermore, monosaccharide compositional analysis showed that Dps contained mannose, glucose, and an unknown sugar residue. Finally, jacalin-binding Dps was detected in larger amounts during the bacterial earlier growth periods, whereas high detection of total Dps was verified throughout the bacterial growth period.ConclusionTaken together, these results indicate that Dps undergoes post-translational modifications in the pre- and early stationary phases of bacterial growth. There is also evidence that a small mannose-containing oligosaccharide is linked to this bacterial protein.

Hematophagous insects are vectors of diseases that affect hundreds of millions of people worldwide. A common physiological event in the life of these insects is the hydrolysis of host hemoglobin in the digestive tract, leading to a massive release of heme, a known prooxidant molecule. Diverse organisms, from bacteria to plants, express the enzyme heme oxygenase, which catalyzes the oxidative degradation of heme to biliverdin (BV) IX, CO, and iron. Here, we show that the kissing bug Rhodnius prolixus , a vector of Chagas' disease, has a unique heme-degradation pathway wherein heme is first modified by addition of two cysteinylglycine residues before cleavage of the porphyrin ring, followed by trimming of the dipeptides. Furthermore, in contrast to most known heme oxygenases, which generate BV IXα, in this insect, the end product of heme detoxification is a dicysteinyl-BV IXγ. Based on these results, we propose a heme metabolizing pathway that includes the identified intermediates produced during modification and cleavage of the heme porphyrin ring.