Extracellular Vesicles from Trypanosoma brucei Mediate Virulence Factor Transfer and Cause Host Anemia.

Intercellular communication between parasites and with host cells provides mechanisms for parasite development, immune evasion, and disease pathology. Bloodstream African trypanosomes produce membranous nanotubes that originate from the flagellar membrane and disassociate into free extracellular vesicles (EVs). Trypanosome EVs contain several flagellar proteins that contribute to virulence, and Trypanosoma brucei rhodesiense EVs contain the serum resistance-associated protein (SRA) necessary for human infectivity. T. b. rhodesiense EVs transfer SRA to non-human infectious trypanosomes, allowing evasion of human innate immunity. Trypanosome EVs can also fuse with mammalian erythrocytes, resulting in rapid erythrocyte clearance and anemia. These data indicate that trypanosome EVs are organelles mediating non-hereditary virulence factor transfer and causing host erythrocyte remodeling, inducing anemia.

The Trypanosoma brucei MISP family of invariant proteins is co-expressed with BARP as triple helical bundle structures on the surface of salivary gland forms, but is dispensable for parasite development within the tsetse vector.

Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomic analyses of saliva from T. brucei-infected tsetse flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of glycosylphosphatidylinositol (GPI)-anchored surface proteins herein named as Metacyclic Invariant Surface Proteins (MISP) because of its predominant expression on the surface of metacyclic trypomastigotes. The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are potentially extended above the metacyclic VSG coat, and thus could be tested as a transmission-blocking vaccine target. However, vaccination with recombinant MISP360 isoform did not protect mice against a T. brucei infectious tsetse bite. Lastly, both CRISPR-Cas9-driven knock out and RNAi knock down of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We suggest MISP may be relevant during trypanosome transmission or establishment in the vertebrate's skin.

Fatty acid elongases 1-3 have distinct roles in mitochondrial function, growth, and lipid homeostasis in Trypanosoma cruzi.

Trypanosomatids are a diverse group of uniflagellate protozoan parasites that include globally relevant pathogens such as Trypanosoma cruzi, the causative agent of Chagas disease. Trypanosomes lack the fatty acid synthase system typically used for de novo fatty acid (FA) synthesis in other eukaryotes. Instead, these microbes have evolved a modular FA elongase (ELO) system comprised of individual ELO enzymes (ELO1-4) that can operate processively to generate long chain- and very long chain-FAs. The importance of ELO's for maintaining lipid homeostasis in trypanosomatids is currently unclear, given their ability to take up and utilize exogenous FAs for lipid synthesis. To assess ELO function in T. cruzi, we generated individual KO lines, Δelo1, Δelo2, and Δelo3, in which the genes encoding ELO1-3 were functionally disrupted in the parasite insect stage (epimastigote). Using unbiased lipidomic and metabolomic analyses, in combination with metabolic tracing and biochemical approaches, we demonstrate that ELO2 and ELO3 are required for global lipid homeostasis, whereas ELO1 is dispensable for this function. Instead, ELO1 activity is needed to sustain mitochondrial activity and normal growth in T. cruzi epimastigotes. The cross-talk between microsomal ELO1 and the mitochondrion is a novel finding that, we propose, merits further examination of the trypanosomatid ELO pathway as critical for central metabolism.

Circulating Blood Prognostic Biomarker Signatures for Hemorrhagic Cerebral Cavernous Malformations (CCMs).

Cerebral cavernous malformations (CCMs) are a neurological disorder characterized by enlarged intracranial capillaries in the brain, increasing the susceptibility to hemorrhagic strokes, a major cause of death and disability worldwide. The limited treatment options for CCMs underscore the importance of prognostic biomarkers to predict the likelihood of hemorrhagic events, aiding in treatment decisions and identifying potential pharmacological targets. This study aimed to identify blood biomarkers capable of diagnosing and predicting the risk of hemorrhage in CCM1 patients, establishing an initial set of circulating biomarker signatures. By analyzing proteomic profiles from both human and mouse CCM models and conducting pathway enrichment analyses, we compared groups to identify potential blood biomarkers with statistical significance. Specific candidate biomarkers primarily associated with metabolism and blood clotting pathways were identified. These biomarkers show promise as prognostic indicators for CCM1 deficiency and the risk of hemorrhagic stroke, strongly correlating with the likelihood of hemorrhagic cerebral cavernous malformations (CCMs). This lays the groundwork for further investigation into blood biomarkers to assess the risk of hemorrhagic CCMs.

Specific Recognition of ß-Galactofuranose-Containing Glycans of Synthetic Neoglycoproteins by Sera of Chronic Chagas Disease Patients.

Chagas disease (CD) can be accurately diagnosed by detecting Trypanosoma cruzi in patients' blood using polymerase chain reaction (PCR). However, parasite-derived biomarkers are of great interest for the serological diagnosis and early evaluation of chemotherapeutic efficacy when PCR may fail, owing to a blood parasite load below the method's limit of detection. Previously, we focused on the detection of specific anti-α-galactopyranosyl (α-Gal) antibodies in chronic CD (CCD) patients elicited by α-Gal glycotopes copiously expressed on insect-derived and mammal-dwelling infective parasite stages. Nevertheless, these stages also abundantly express cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and glycoinositolphospholipids (GIPLs) bearing nonreducing terminal ß-galactofuranosyl (ß-Galf) residues, which are equally foreign to humans and, therefore, highly immunogenic. Here we report that CCD patients' sera react specifically with synthetic ß-Galf-containing glycans. We took a reversed immunoglycomics approach that entailed: (a) Synthesis of T. cruzi GIPL-derived Galfß1,3Manpα-(CH2)3SH (glycan G29SH) and Galfß1,3Manpα1,2-[Galfß1,3]Manpα-(CH2)3SH (glycan G32SH); and (b) preparation of neoglycoproteins NGP29b and NGP32b, and their evaluation in a chemiluminescent immunoassay. Receiver-operating characteristic analysis revealed that NGP32b can distinguish CCD sera from sera of healthy individuals with 85.3% sensitivity and 100% specificity. This suggests that Galfß1,3Manpα1,2-[Galfß1,3]Manpα is an immunodominant glycotope and that NGP32b could potentially be used as a novel CCD biomarker.

A Branched and Double Alpha-Gal-Bearing Synthetic Neoglycoprotein as a Biomarker for Chagas Disease.

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi and affects 6-7 million people worldwide. The diagnosis is still challenging, due to extensive parasite diversity encompassing seven genotypes (TcI-VI and Tcbat) with diverse ecoepidemiological, biological, and pathological traits. Chemotherapeutic intervention is usually effective but associated with severe adverse events. The development of safer, more effective therapies is hampered by the lack of biomarker(s) (BMKs) for the early assessment of therapeutic outcomes. The mammal-dwelling trypomastigote parasite stage expresses glycosylphosphatidylinositol-anchored mucins (tGPI-MUC), whose O-glycans are mostly branched with terminal, nonreducing α-galactopyranosyl (α-Gal) glycotopes. These are absent in humans, and thus highly immunogenic and inducers of specific CD anti-α-Gal antibodies. In search for α-Gal-based BMKs, here we describe the synthesis of neoglycoprotein NGP11b, comprised of a carrier protein decorated with the branched trisaccharide Galα(1,2)[Galα(1,6)]Galß. By chemiluminescent immunoassay using sera/plasma from chronic CD (CCD) patients from Venezuela and Mexico and healthy controls, NGP11b exhibited sensitivity and specificity similar to that of tGPI-MUC from genotype TcI, predominant in those countries. Preliminary evaluation of CCD patients subjected to chemotherapy showed a significant reduction in anti-α-Gal antibody reactivity to NGP11b. Our data indicated that NGP11b is a potential BMK for diagnosis and treatment assessment in CCD patients.

Plasma-Derived Extracellular Vesicles as Potential Biomarkers in Heart Transplant Patient with Chronic Chagas Disease.

Chagas disease is emerging in countries to which it is not endemic. Biomarkers for earlier therapeutic response assessment in patients with chronic Chagas disease are needed. We profiled plasma-derived extracellular vesicles from a heart transplant patient with chronic Chagas disease and showed the potential of this approach for discovering such biomarkers.

Anti-α-Gal antibodies detected by novel neoglycoproteins as a diagnostic tool for Old World cutaneous leishmaniasis caused by Leishmania major.

Outbreaks of Old World cutaneous leishmaniasis (CL) have significantly increased due to the conflicts in the Middle East, with most of the cases occurring in resource-limited areas such as refugee settlements. The standard methods of diagnosis include microscopy and parasite culture, which have several limitations. To address the growing need for a CL diagnostic that can be field applicable, we have identified five candidate neoglycoproteins (NGPs): Galα (NGP3B), Galα(1,3)Galα (NGP17B), Galα(1,3)Galß (NGP9B), Galα(1,6)[Galα(1,2)]Galß (NGP11B), and Galα(1,3)Galß(1,4)Glcß (NGP1B) that are differentially recognized in sera from individuals with Leishmania major infection as compared with sera from heterologous controls. These candidates contain terminal, non-reducing α-galactopyranosyl (α-Gal) residues, which are known potent immunogens to humans. Logistic regression models found that NGP3B retained the best diagnostic potential (area under the curve from receiver-operating characteristic curve = 0.8). Our data add to the growing body of work demonstrating the exploitability of the human anti-α-Gal response in CL diagnosis.

Enhancing glycan isomer separations with metal ions and positive and negative polarity ion mobility spectrometry-mass spectrometry analyses.

Glycomics has become an increasingly important field of research since glycans play critical roles in biology processes ranging from molecular recognition and signaling to cellular communication. Glycans often conjugate with other biomolecules, such as proteins and lipids, and alter their properties and functions, so glycan characterization is essential for understanding the effects they have on cellular systems. However, the analysis of glycans is extremely difficult due to their complexity and structural diversity (i.e., the number and identity of monomer units, and configuration of their glycosidic linkages and connectivities). In this work, we coupled ion mobility spectrometry with mass spectrometry (IMS-MS) to characterize glycan standards and biologically important isomers of synthetic αGal-containing O-glycans including glycotopes of the protozoan parasite Trypanosoma cruzi, which is the causative agent of Chagas disease. IMS-MS results showed significant differences for the glycan structural isomers when analyzed in positive and negative polarity and complexed with different metal cations. These results suggest that specific metal ions or ion polarities could be used to target and baseline separate glycan isomers of interest with IMS-MS. Graphical abstract Glycan isomers, such as fructose and glucose, show distinct separations in positive and negative ion mode.

Synthesis of Galα(1,3)Galß(1,4)GlcNAcα-, Galß(1,4)GlcNAcα- and GlcNAc-containing neoglycoproteins and their immunological evaluation in the context of Chagas disease.

The protozoan parasite, Trypanosoma cruzi, the etiologic agent of Chagas disease (ChD), has a cell surface covered by immunogenic glycoconjugates. One of the immunodominant glycotopes, the trisaccharide Galα(1,3)Galß(1,4)GlcNAcα, is expressed on glycosylphosphatidylinositol-anchored mucins of the infective trypomastigote stage of T. cruzi and triggers high levels of protective anti-α-Gal antibodies (Abs) in infected individuals. Here, we have efficiently synthesized the mercaptopropyl glycoside of that glycotope and conjugated it to maleimide-derivatized bovine serum albumin (BSA). Chemiluminescent-enzyme-linked immunosorbent assay revealed that Galα(1,3)Galß(1,4)GlcNAcα-BSA is recognized by purified anti-α-Gal Abs from chronic ChD patients ∼230-fold more strongly than by anti-α-Gal Abs from sera of healthy individuals (NHS anti-α-Gal). Similarly, the pooled sera of chronic Chagas disease patients (ChHSP) recognized Galα(1,3)Galß(1,4)GlcNAcα ∼20-fold more strongly than pooled NHS. In contrast, the underlying disaccharide Galß(1,4)GlcNAcα and the monosaccharide GlcNAcα or GlcNAcß conjugated to BSA are poorly or not recognized by purified anti-α-Gal Abs or sera from Chagasic patients or healthy individuals. Our results highlight the importance of the terminal Galα moiety for recognition by Ch anti-α-Gal Abs and the lack of Abs against nonself Galß(1,4)GlcNAcα and GlcNAcα glycotopes. The substantial difference in binding of Ch vs. NHS anti-α-Gal Abs to Galα(1,3)Galß(1,4)GlcNAcα-BSA suggests that this neoglycoprotein (NGP) might be suitable for experimental vaccination. To this end, the Galα(1,3)Galß(1,4)GlcNAcα-BSA NGP was then used to immunize α1,3-galactosyltransferase-knockout mice, which produced antibody titers 40-fold higher as compared with pre-immunization titers. Taken together, our results indicate that the synthetic Galα(1,3)Galß(1,4)GlcNAcα glycotope coupled to a carrier protein could be a potential diagnostic and vaccine candidate for ChD.

Compositional and immunobiological analyses of extracellular vesicles released by Candida albicans.

The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transport mechanism to trans-cell wall passage of macromolecules. Previous studies have revealed the presence of EV in culture supernatants from fungal pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Malassezia sympodialis and Candida albicans. Here we investigated the size, composition, kinetics of internalization by bone marrow-derived murine macrophages (MO) and dendritic cells (DC), and the immunomodulatory activity of C. albicans EV. We also evaluated the impact of EV on fungal virulence using the Galleria mellonella larvae model. By transmission electron microscopy and dynamic light scattering, we identified two populations ranging from 50 to 100 nm and 350 to 850 nm. Two predominant seroreactive proteins (27 kDa and 37 kDa) and a group of polydispersed mannoproteins were observed in EV by immunoblotting analysis. Proteomic analysis of C. albicans EV revealed proteins related to pathogenesis, cell organization, carbohydrate and lipid metabolism, response to stress, and several other functions. The major lipids detected by thin-layer chromatography were ergosterol, lanosterol and glucosylceramide. Short exposure of MO to EV resulted in internalization of these vesicles and production of nitric oxide, interleukin (IL)-12, transforming growth factor-beta (TGF-ß) and IL-10. Similarly, EV-treated DC produced IL-12p40, IL-10 and tumour necrosis factor-alpha. In addition, EV treatment induced the up-regulation of CD86 and major histocompatibility complex class-II (MHC-II). Inoculation of G. mellonella larvae with EV followed by challenge with C. albicans reduced the number of recovered viable yeasts in comparison with infected larvae control. Taken together, our results demonstrate that C. albicans EV were immunologically active and could potentially interfere with the host responses in the setting of invasive candidiasis.

Characterization of Lipids and Proteins Associated to the Cell Wall of the Acapsular Mutant Cryptococcus neoformans Cap 67.

Cryptococcus neoformans is an opportunistic human pathogen that causes life-threatening meningitis. In this fungus, the cell wall is exceptionally not the outermost structure due to the presence of a surrounding polysaccharide capsule, which has been highly studied. Considering that there is little information about C. neoformans cell wall composition, we aimed at describing proteins and lipids extractable from this organelle, using as model the acapsular mutant C. neoformans cap 67. Purified cell wall preparations were extracted with either chloroform/methanol or hot sodium dodecyl sulfate. Total lipids fractionated in silica gel 60 were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS), while trypsin digested proteins were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We detected 25 phospholipid species among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Two glycolipid species were identified as monohexosyl ceramides. We identified 192 noncovalently linked proteins belonging to different metabolic processes. Most proteins were classified as secretory, mainly via nonclassical mechanisms, suggesting a role for extracellular vesicles (EV) in transwall transportation. In concert with that, orthologs from 86% of these proteins have previously been reported both in fungal cell wall and/or in EV. The possible role of the presently described structures in fungal-host relationship is discussed.

Environmental Effects of Nanoceria on Seed Production of Common Bean (Phaseolus vulgaris): A Proteomic Analysis.

The rapidly growing literature on the response of edible plants to nanoceria has provided evidence of its uptake and bioaccumulation, which delineates a possible route of entry into the food chain. However, little is known about how the residing organic matter in soil may affect the bioavailability and resulting impacts of nanoceria on plants. Here, we examined the effect of nanoceria exposure (62.5-500 mg/kg) on kidney bean (Phaseolus vulgaris) productivity and seed quality as a function of soil organic matter content. Cerium accumulation in the seeds produced from plants in organic matter enriched soil showed a dose-dependent increase, unlike in low organic matter soil treatments. Seeds obtained upon nanoceria exposure in soils with higher organic matter were more susceptible to changes in nutrient quality. A quantitative proteomic analysis of the seeds produced upon nanoceria exposure provided evidence for upregulation of stress-related proteins at 62.5 and 125 mg/kg nanoceria treatments. Although the plants did not exhibit overt toxicity, the major seed proteins primarily associated with nutrient storage (phaseolin) and carbohydrate metabolism (lectins) were significantly down-regulated in a dose dependent manner upon nanoceria exposure. This study thus suggests that nanoceria exposures may negatively affect the nutritional quality of kidney beans at the cellular and molecular level. More confirmatory studies with nanoceria along different species using alternative and orthogonal "omic" tools are currently under active investigation, which will enable the identification of biomarkers of exposure and susceptibility.

Role of the Apt1 protein in polysaccharide secretion by Cryptococcus neoformans.

Flippases are key regulators of membrane asymmetry and secretory mechanisms. Vesicular polysaccharide secretion is essential for the pathogenic mechanisms of Cryptococcus neoformans. On the basis of the observations that flippases are required for polysaccharide secretion in plants and the putative Apt1 flippase is required for cryptococcal virulence, we analyzed the role of this enzyme in polysaccharide release by C. neoformans, using a previously characterized apt1Δ mutant. Mutant and wild-type (WT) cells shared important phenotypic characteristics, including capsule morphology and dimensions, glucuronoxylomannan (GXM) composition, molecular size, and serological properties. The apt1Δ mutant, however, produced extracellular vesicles (EVs) with a lower GXM content and different size distribution in comparison with those of WT cells. Our data also suggested a defective intracellular GXM synthesis in mutant cells, in addition to changes in the architecture of the Golgi apparatus. These findings were correlated with diminished GXM production during in vitro growth, macrophage infection, and lung colonization. This phenotype was associated with decreased survival of the mutant in the lungs of infected mice, reduced induction of interleukin-6 (IL-6) cytokine levels, and inefficacy in colonization of the brain. Taken together, our results indicate that the lack of APT1 caused defects in both GXM synthesis and vesicular export to the extracellular milieu by C. neoformans via processes that are apparently related to the pathogenic mechanisms used by this fungus during animal infection.

Glucosylceramide transferase activity is critical for encystation and viable cyst production by an intestinal protozoan, Giardia lamblia.

The production of viable cysts by Giardia is essential for its survival in the environment and for spreading the infection via contaminated food and water. The hallmark of cyst production (also known as encystation) is the biogenesis of encystation-specific vesicles (ESVs) that transport cyst wall proteins to the plasma membrane of the trophozoite before laying down the protective cyst wall. However, the molecules that regulate ESV biogenesis and maintain cyst viability have never before been identified. Here, we report that giardial glucosylceramide transferase-1 (gGlcT1), an enzyme of sphingolipid biosynthesis, plays a key role in ESV biogenesis and maintaining cyst viability. We find that overexpression of this enzyme induced the formation of aggregated/enlarged ESVs and generated clustered cysts with reduced viability. The silencing of gGlcT1 synthesis by antisense morpholino oligonucleotide abolished ESV production and generated mostly nonviable cysts. Interestingly, when gGlcT1-overexpressed Giardia was transfected with anti-gGlcT1 morpholino, the enzyme activity, vesicle biogenesis, and cyst viability returned to normal, suggesting that the regulated expression of gGlcT1 is important for encystation and viable cyst production. Furthermore, the overexpression of gGlcT1 increased the influx of membrane lipids and fatty acids without altering the fluidity of plasma membranes, indicating that the expression of gGlcT1 activity is linked to lipid internalization and maintaining the overall lipid balance in this parasite. Taken together, our results suggest that gGlcT1 is a key player of ESV biogenesis and cyst viability and therefore could be targeted for developing new anti-giardial therapies.

Detection of high levels of anti-α-galactosyl antibodies in sera of patients with Old World cutaneous leishmaniasis: a possible tool for diagnosis and biomarker for cure in an elimination setting.

In the Kingdom of Saudi Arabia (KSA), Old World cutaneous leishmaniasis (CL) is mainly caused by Leishmania major and Leishmania tropica parasites. Diagnosis of CL is predominately made by clinicians, who at times fail to detect the disease and are unable to identify parasite species. Here, we report the development of a chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) to measure the levels of anti-α-galactosyl antibodies in human sera. Using this assay, we have found that individuals infected with either Leishmania spp. had significantly elevated levels (up to 9-fold higher) of anti-α-Gal IgG compared to healthy control individuals. The assay sensitivity was 96% for L. major (95% CI; 94-98%) and 91% for L. tropica (95% CI; 86-98%) infections and therefore equivalent to restriction fragment length polymorphism-polymerase chain reaction analysis of parasite ITS1 gene. In addition, the assay had higher sensitivity than microscopy analysis, which only detected 68 and 45% of the L. major and L. tropica infections, respectively. Interestingly, up to 2 years following confirmed CL cure individuals had 28-fold higher levels of anti-α-Gal IgG compared to healthy volunteers. Monitoring levels of anti-α-Gal antibodies can be exploited as both a diagnostic tool and as a biomarker of cure of Old World CL in disease elimination settings.

Big is not better: Comparing two alpha-Gal-bearing glycotopes in neoglycoproteins as biomarkers for Leishmania (Viannia) braziliensis infection.

The protozoan parasite Leishmania (Viannia) braziliensis is among Latin America's most widespread Leishmania species and is responsible for tegumentary leishmaniasis (TL). This disease has multiple clinical presentations, with cutaneous leishmaniasis (CL) being the most frequent. It manifests as one or a few localized skin ulcers, which can spread to other body areas. Hence, early diagnosis and treatment, typically with pentavalent antimonials, is critical. Traditional diagnostic methods, like parasite culture, microscopy, or the polymerase chain reaction (PCR) for detection of the parasite DNA, have limitations due to the uneven distribution of parasites in biopsy samples. Nonetheless, studies have revealed high levels of parasite-specific anti-α-Gal antibodies in L. (V.) braziliensis-infected patients. Previously, we demonstrated that the neoglycoprotein NGP28b, consisting of the L. (Leishmania) major type-2 glycoinositolphospholipid (GIPL)-3-derived trisaccharide Galpα1,6Galpα1,3Galfß conjugated to bovine serum albumin (BSA) via a linker, acts as a reliable serological biomarker (BMK) for L. (V.) braziliensis infection in Brazil. This indicates the presence of GIPL-3 or a similar structure in this parasite, and its terminal trisaccharide either functions as or is part of an immunodominant glycotope. Here, we explored whether extending the trisaccharide with a mannose unit would enhance its efficacy as a biomarker for the serological detection of L. (V.) braziliensis. We synthesized the tetrasaccharide Galpα1,6Galpα1,3Galfß1,3Manpα(CH2)3SH (G31SH) and conjugated it to maleimide-functionalized BSA to afford NGP31b. When we assessed the efficacy of NGP28b and NGP31b by chemiluminescent enzyme-linked immunosorbent assay on a cohort of CL patients with L. (V.) braziliensis infection from Bolivia and Argentina against a healthy control group, both NGPs exhibited similar or identical sensitivity, specificity, and accuracy. This finding implies that the mannose moiety at the reducing end is not part of the glycotope recognized by the parasite-specific anti-α-Gal antibodies in patients' sera, nor does it exert a relevant influence on the terminal trisaccharide's conformation. Moreover, the mannose does not seem to inhibit glycan-antibody interactions. Therefore, NGP31b is a viable and dependable BMK for the serodiagnosis of CL caused by L. (V.) braziliensis.

Using immunoproteomics to identify alpha-enolase as an autoantigen in liver fibrosis.

Liver fibrosis results from extracellular matrix accumulation during the wound healing process when the liver is insulted with chronic viral infection, inflammation, or alcoholic diseases. The current diagnosis of liver fibrosis is mainly dependent on biopsy, which is an invasive approach. Identification of serological biomarkers has been considered as the most promising way for early detection of the disease. Although several biomarkers in liver fibrosis have been identified, the problem is that these markers can be also detected in fibrogenesis that occurred in other organs. In this study, we have identified and characterized some cellular proteins that can be recognized by autoantibodies in the sera from patients with precirrhotic stage of liver fibrosis. Among 180 sera from patients with liver fibrosis, 14.4% (26/180) of sera contained autoantibody against a protein migrating around 47 kDa on SDS-PAGE gel. Indirect immunofluorescence assay using purified autoantibody against the 47-kDa protein showed that this protein mainly localized in the cytoplasm. Using immunoproteomic approach, the 47-kDa protein was identified as alpha-enolase. In further study, the frequency of antialpha-enolase antibody in sera from patients with precirrhotic stage of liver fibrosis (21.6%, 27/125) was significantly higher than that in sera from patients with cirrhosis (9.1%, 5/55) and liver cancer (14.3%, 12/84), as well as in sera from healthy individuals (4.1%, 3/74). Therefore, alpha-enolase is an autoantigen that elicits autoimmune response in liver fibrosis and can be a potential prognostic factor for liver fibrosis diagnosis.

Proteomic analysis of Trypanosoma cruzi secretome: characterization of two populations of extracellular vesicles and soluble proteins.

Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in Trypanosoma cruzi; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that T. cruzi releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes, the second enriched in smaller vesicles resembling exosomes, and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that T. cruzi uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells.

Immunomodulatory and antibacterial effects of cystatin 9 against Francisella tularensis.

Cystatin 9 (CST9) is a member of the type 2 cysteine protease inhibitor family, which has been shown to have immunomodulatory effects that restrain inflammation, but its functions against bacterial infections are unknown. Here, we report that purified human recombinant (r)CST9 protects against the deadly bacterium Francisella tularensis (Ft) in vitro and in vivo. Macrophages infected with the Ft human pathogen Schu 4 (S4), then given 50 pg of rCST9 exhibited significantly decreased intracellular bacterial replication and increased killing via preventing the escape of S4 from the phagosome. Further, rCST9 induced autophagy in macrophages via the regulation of the mammalian target of rapamycin (mTOR) signaling pathways. rCST9 promoted the upregulation of macrophage proteins involved in antiinflammation and antiapoptosis, while restraining proinflammatory-associated proteins. Interestingly, the viability and virulence of S4 also was decreased directly by rCST9. In a mouse model of Ft inhalation, rCST9 significantly decreased organ bacterial burden and improved survival, which was not accompanied by excessive cytokine secretion or subsequent immune cell migration. The current report is the first to show the immunomodulatory and antimicrobial functions of rCST9 against Ft. We hypothesize that the attenuation of inflammation by rCST9 may be exploited for therapeutic purposes during infection.