vSEMERA: pilot project assessing health profession students' experiences in an international virtual research program.

BACKGROUND: The "Virtual Semester for Medical Research Aachen" (vSEMERA) is an international, interdisciplinary, virtual education program developed for health profession students. The first edition (2021) was hosted by the Medical Faculty of RWTH Aachen University (Germany) in cooperation with Centro Universitário Christus (Brazil) and Universidad Peruana Cayetano Heredia (Peru). The primary aim of the 12-weeks program was to provide students with skills in health science research and prepare them for scientific career paths. METHODS: vSEMERA was built on a virtual learning platform, the "vSEMERA-Campus", designed to foster students' learning process and social interactions. Maximum flexibility was offered through synchronous and asynchronous teaching, enabling participants to join via any device from any part of the Globe alongside their regular studies. For the program's first edition (September - November 2021), health profession students from Germany, Brazil, Peru, Spain, and Italy filled all 30 available spots. Satisfaction, quality of the program and courses offered, as well as perceived learning outcomes, were examined using questionnaires throughout and at the end of the program. RESULTS: The program received a rating of 4.38 out of 5 stars. While it met most expectations (4.29 out of 5), participants were unable to attend as many courses as intended (2.81 out of 5), mainly due to scheduling conflicts with the home university schedule (46%), internships (23%), and general timing issues (31%). Participants acknowledged considerable improvements in their scientific skills, English language skills, confidence in scientific project management, research career progression, and enthusiasm for a scientific career. CONCLUSIONS: vSEMERA represents a promising example of an online international learning and exchange program using pedagogical and technological elements of virtual collaboration and teaching. In addition to advancing future vSEMERA editions, our results may offer insights for similar projects that address the targeted integration of scientific research education into an international, digital learning environment.

Increased hypoxic proliferative response and gene expression in erythroid progenitor cells of Andean highlanders with chronic mountain sickness.

Excessive erythrocytosis (EE) is the main sign of chronic mountain sickness (CMS), a maladaptive clinical syndrome prevalent in Andean and other high-altitude populations worldwide. The pathophysiological mechanism of EE is still controversial, as physiological variability of systemic respiratory, cardiovascular, and hormonal responses to chronic hypoxemia complicates the identification of underlying causes. Induced pluripotent stem cells derived from CMS highlanders showed increased expression of genes relevant to the regulation of erythropoiesis, angiogenesis, cardiovascular, and steroid-hormone function that appear to explain the exaggerated erythropoietic response. However, the cellular response to hypoxia in native CMS cells is yet unknown. This study had three related aims: to determine the hypoxic proliferation of native erythroid progenitor burst-forming unit-erythroid (BFU-E) cells derived from CMS and non-CMS peripheral blood mononuclear cells; to examine their sentrin-specific protease 1 (SENP1), GATA-binding factor 1 (GATA1), erythropoietin (EPO), and EPO receptor (EPOR) expression; and to investigate the functional upstream role of SENP1 in native progenitor differentiation into erythroid precursors. Native CMS BFU-E colonies showed increased proliferation under hypoxic conditions compared with non-CMS cells, together with an upregulated expression of SENP1, GATA1, EPOR; and no difference in EPO expression. Knock-down of the SENP1 gene abolished the augmented proliferative response. Thus, we demonstrate that native CMS progenitor cells produce a larger proportion of erythroid precursors under hypoxia and that SENP1 is essential for proliferation. Our findings suggest a significant intrinsic component for developing EE in CMS highlanders at the cellular and gene expression level that could be further enhanced by systemic factors such as alterations in respiratory control, or differential hormonal patterns.

TNF-α blockade suppresses pericystic inflammation following anthelmintic treatment in porcine neurocysticercosis.

BACKGROUND: Neurocysticercosis (NCC) is an infection of the brain with the larval cyst of the tapeworm, Taenia solium. Cysticidal treatment induces parasite killing resulting in a post inflammatory response and seizures, which generally requires corticosteroid treatment to control inflammation. The nature of this response and how to best control it is unclear. We investigated the anti-inflammatory effects of pretreatment with etanercept (ETN), an anti-tumor necrosis factor agent, or dexamethasone (DEX), a high potency corticosteroid, on the post treatment inflammatory response in naturally infected pigs with neurocysticercosis after a single dose of the cysticidal drug praziquantel (PZQ). METHODOLOGY/PRINCIPAL FINDINGS: We followed the methods from a previously developed treatment model of NCC in naturally infected swine. The four study groups of infected pigs included 3 groups treated with PZQ on day 0: PZQ-treated alone (100 mg/kg PO; n = 9), pretreated with dexamethasone (DEX, 0.2 mg/kg IM administered on days -1, +1 and +3; n = 6), and pretreated with etanercept (ETN, 25 mg IM per animal on days -7 and 0; n = 6). The fourth group remained untreated (n = 3). As measured by quantitative RT-PCR, ETN pretreatment depressed transcription of a wide range of proinflammatory, regulatory and matrix protease encoding genes at 120 hr post PZQ treatment in capsules of cysts that demonstrated extravasated Evans Blue (EB) (a measure of blood brain barrier dysfunction) compared to animals not receiving ETN. Transcription was significantly depressed for the proinflammatory genes tumor necrosis factor (TNF)-α, and interferon (IFN)-γ; the inflammation regulating genes cytotoxic T-lymphocyte-associated protein (CTLA)4, interleukin (IL)-13 and transforming growth factor (TGF)-ß; the tissue remodeling genes matrix metalloprotease (MMP)1 and 9, tissue inhibitors of metalloproteases (TIMP)1 and 2, and the genes regulating endothelial function vascular endothelial growth factor (VEGF)1, angiopoietin (Ang)1, Ang 2, and platelet endothelial cell adhesion molecule (PECAM)-1. In contrast, transcription was only modestly decreased in the DEX pretreated pigs compared to PZQ alone, and only for TNF-α, IL-6, IFN-γ, TGF-ß and Ang1. IL-10 was not affected by either ETN or DEX pretreatments. The degree of inflammation, assessed by semi-quantitative inflammatory scores, was modestly decreased in both ETN and DEX pretreated animals compared to PZQ treated pigs whereas cyst damage scores were moderately decreased only in cysts from DEX pretreated pigs. However, the proportion of cysts with EB extravasation was not significantly changed in ETN and DEX pretreated groups. CONCLUSIONS/SIGNIFICANCE: Overall, TNF-α blockade using ETN treatment modulated expression of a large variety of genes that play a role in induction and control of inflammation and structural changes. In contrast the number of inflammatory cells was only moderately decreased suggesting weaker effects on cell migration into the inflammatory capsules surrounding cysts than on release of modulatory molecules. Taken together, these data suggest that TNF-α blockade may provide a viable strategy to manage post-treatment pericystic inflammation that follows antiparasitic therapy for neurocysticercosis.

MicroRNAs in Taenia solium Neurocysticercosis: Insights as Promising Agents in Host-Parasite Interaction and Their Potential as Biomarkers.

MicroRNAs (miRNAs) are short, endogenous, non-coding, single-stranded RNAs involved in post-transcriptional gene regulation. Although, several miRNAs have been identified in parasitic helminths, there is little information about their identification and function in Taenia. Furthermore, the impact of miRNAs in neurocysticercosis, the brain infection caused by larvae of Taenia solium is still unknown. During chronic infection, T. solium may activate numerous mechanisms aimed to modulate host immune responses. Helminthic miRNAs might also have effects on host mRNA expression and thus play an important role regulating host-parasite interactions. Also, the diagnosis of this disease is difficult and it usually requires neuroimaging and confirmatory serology. Since miRNAs are stable when released, they can be detected in body fluids and therefore have potential to diagnose infection, determine parasite burden, and ascertain effectiveness of treatment or disease progression, for instance. This review discusses the potential roles of miRNAs in T. solium infection, including regulation of host-parasite relationships and their eventual use as diagnostic or disease biomarkers. Additionally, we summarize the bioinformatics resources available for identification of T. solium miRNAs and prediction of their targets.

Radiological evolution of porcine neurocysticercosis after combined antiparasitic treatment with praziquantel and albendazole.

BACKGROUND: The onset of anthelmintic treatment of neurocysticercosis (NCC) provokes an acute immune response of the host, which in human cases is associated with exacerbation of neurological symptoms. This inflammation can occur at the first days of therapy. So, changes in the brain cysts appearance may be detected by medical imaging. We evaluated radiological changes in the appearance of brain cysts (enhancement and size) on days two and five after the onset of antiparasitic treatment using naturally infected pigs as a model for human NCC. METHODS AND RESULTS: Contrast T1-weighted magnetic resonance imaging with gadolinium was performed before and after antiparasitic treatment. Eight NCC-infected pigs were treated with praziquantel plus albendazole and euthanized two (n = 4) and five (n = 4) days after treatment; another group of four infected pigs served as untreated controls. For each lesion, gadolinium enhancement intensity (GEI) and cyst volume were measured at baseline and after antiparasitic treatment. Volume and GEI quantification ratios (post/pre-treatment measures) were used to appraise the effect of treatment. Cysts from untreated pigs showed little variations between their basal and post treatment measures. At days 2 and 5 there were significant increases in GEI ratio compared with the untreated group (1.32 and 1.47 vs 1.01, p = 0.021 and p = 0.021). Cyst volume ratios were significantly lower at days 2 and 5 compared with the untreated group (0.60 and 0.22 vs 0.95, p = 0.04 and p = 0.02). Cysts with lower cyst volume ratios showed more marked post-treatment inflammation, loss of vesicular fluid and cyst wall wrinkling. CONCLUSION/SIGNIFICANCE: A significant and drastic reduction of cyst size and increased pericystic enhancement occur in the initial days after antiparasitic treatment as an effect of acute perilesional immune response. These significant changes showed that early anthelmintic efficacy (day two) can be detected using magnetic resonance imaging.

Perilesional Inflammation in Neurocysticercosis - Relationship Between Contrast-Enhanced Magnetic Resonance Imaging, Evans Blue Staining and Histopathology in the Pig Model.

BACKGROUND: Disease manifestations in neurocysticercosis (NCC) are frequently due to inflammation of degenerating Taenia solium brain cysts. Exacerbated inflammation post anthelmintic treatment is associated with leakage of the blood brain barrier (BBB) using Evans blue (EB) staining. How well EB extravasation into the brain correlates with magnetic resonance imaging (MRI) using gadolinium (Gd) enhancement as a contrast agent and pericystic inflammation was analyzed in pigs harboring brain cysts of Taenia solium. METHODOLOGY/PRINCIPAL FINDINGS: Three groups of 4 naturally infected pigs were assessed. The first and second groups were treated with both praziquantel plus albendazole and sacrificed two and five days post treatment, respectively. A third untreated group remained untreated. Pigs were injected with EB two hours prior to evaluation by Gd-enhanced T1-MRI, and euthanized. The EB staining for each cyst capsule was scored (EB grades were 0: 0%; 1: up to 50%; 2: over 50% but less than 100%; 3: 100%). Similarly, the Gd enhancement around each cyst was qualitatively and quantitatively scored from the MRI. The extent of pericystic inflammation on histology was scored in increasing severity as IS1, IS2, IS3 and IS4. Grade 3 EB staining and enhancement was only seen in treated capsules. Also, treated groups had higher Gd intensity than the untreated group. Grades of enhancement correlated significantly with Gd enhancement intensity. EB staining was correlated with Gd enhancement intensity and with IS4 in the treated groups. These correlations were stronger in internally located cysts compared to superficial cysts in treated groups. SIGNIFICANCE: EB staining and Gd enhancement strongly correlate. The intensity of enhancement determined by MRI is a good indication of the degree of inflammation. Similarly, EB staining highly correlates with the degree of inflammation and may be applied to study inflammation in the pig model of NCC.

Anti-Taenia solium monoclonal antibodies for the detection of parasite antigens in body fluids from patients with neurocysticercosis.

Neurocysticercosis (NCC), an infection of the brain by Taenia solium (Ts) cysts, is the most common cause of adult-onset epilepsy in developing countries. Serological testing consists primarily of varying methods to detect antibodies in body fluids and more recently antigen (Ag) detection assays to identify individuals or animals with viable parasites. Antigen assays currently in use employ monoclonal antibodies (mAbs) raised against T. saginata, which have known cross reactivity to animal cestodes but are highly specific in human samples. We produced, characterized and tested 21 mAbs raised against T. solium whole cyst antigens, vesicular fluid or excretory secretory products. Reactivity of the TsmAbs against specific cyst structures was determined using immunofluorescence and immunohistochemistry on histological sections of Ts muscle cysts. Four TsmAbs reacted to vesicular space alone, 9 to the neck and cyst wall, one to the neck and vesicular space and 7 to the neck, cyst wall and vesicular space. An in-house ELISA assay to detect circulating Ts antigen, using the TsmAbs as capture antibodies and a rabbit polyclonal anti-Ts whole cyst antibody as a detector antibody demonstrated that eight of the 21 TsmAbs detected antigens in known NCC-positive human sera and three of these also in urine samples. Reactivity was expressed as normalized ratios of optical densities (OD positive control/OD negative control). Three TsmAbs had ratios >10 and five between 2 and 10. The TsmAbs have potential utility for the diagnosis and post-treatment monitoring of patients with viable NCC infections.

Inflammation Caused by Praziquantel Treatment Depends on the Location of the Taenia solium Cysticercus in Porcine Neurocysticercosis.

BACKGROUND: Neurocysticercosis (NCC), infection of the central nervous system by Taenia solium cysticerci, is a pleomorphic disease. Inflammation around cysticerci is the major cause of disease but is variably present. One factor modulating the inflammatory responses may be the location and characteristics of the brain tissue adjacent to cysticerci. We analyzed and compared the inflammatory responses to cysticerci located in the parenchyma to those in the meninges or cysticerci partially in contact with both the parenchyma and the meninges (corticomeningeal). METHODOLOGY/PRINCIPAL FINDINGS: Histological specimens of brain cysticerci (n = 196) from 11 pigs naturally infected with Taenia solium cysticerci were used. Four pigs were sacrificed after 2 days and four after 5 days of a single dose of praziquantel; 3 pigs did not receive treatment. All pigs were intravenously injected with Evans Blue to assess disruption of the blood-brain barrier. The degree of inflammation was estimated by use of a histological score (ISC) based on the extent of the inflammation in the pericystic areas as assessed in an image composed of several photomicrographs taken at 40X amplification. Parenchymal cysticerci provoked a significantly greater level of pericystic inflammation (higher ISC) after antiparasitic treatment compared to meningeal and corticomeningeal cysticerci. ISC of meningeal cysticerci was not significantly affected by treatment. In corticomeningeal cysticerci, the increase in ISC score was correlated to the extent of the cysticercus adjacent to the brain parenchyma. Disruption of the blood-brain barrier was associated with treatment only in parenchymal tissue. SIGNIFICANCE: Inflammatory response to cysticerci located in the meninges was significantly decreased compared to parenchymal cysticerci. The suboptimal inflammatory response to cysticidal drugs may be the reason subarachnoid NCC is generally refractory to treatment compared to parenchymal NCC.

Post-treatment vascular leakage and inflammatory responses around brain cysts in porcine neurocysticercosis.

Cysticidal treatment of neurocysticercosis, an infection of humans and pig brains with Taenia solium, results in an early inflammatory response directed to cysts causing seizures and focal neurological manifestations. Treatment-induced pericystic inflammation and its association with blood brain barrier (BBB) dysfunction, as determined by Evans blue (EB) extravasation, was studied in infected untreated and anthelmintic-treated pigs. We compared the magnitude and extent of the pericystic inflammation, presence of EB-stained capsules, the level of damage to the parasite, expression of genes for proinflammatory and regulatory cytokines, chemokines, and tissue remodeling by quantitative PCR assays between treated and untreated infected pigs and between EB-stained (blue) and non stained (clear) cysts. Inflammatory scores were higher in pericystic tissues from EB-stained cysts compared to clear cysts from untreated pigs and also from anthelmintic-treated pigs 48 hr and 120 hr after treatment. The degree of inflammation correlated with the severity of cyst wall damage and both increased significantly at 120 hours. Expression levels of the proinflammatory genes for IL-6, IFN-γ, TNF-α were higher in EB-stained cysts compared to clear cysts and unaffected brain tissues, and were generally highest at 120 hr. Additionally, expression of some markers of immunoregulatory activity (IL-10, IL-2Rα) were decreased in EB-stained capsules. An increase in other markers for regulatory T cells (CTLA4, FoxP3) was found, as well as significant increases in expression of two metalloproteases, MMP1 and MMP2 at 48 hr and 120 hr post-treatment. We conclude that the increase in severity of the inflammation caused by treatment is accompanied by both a proinflammatory and a complex regulatory response, largely limited to pericystic tissues with compromised vascular integrity. Because treatment induced inflammation occurs in porcine NCC similar to that in human cases, this model can be used to investigate mechanisms involved in host damaging inflammatory responses and agents or modalities that may control damaging post treatment inflammation.

Identificación de células proliferativas en quistes de Taenia solium / Identification of proliferating cells in Taenia solium cysts

Los neoblastos son células totipotentes, únicas responsables de la proliferación y maduración de tejidos en platelmintos de vida libre. Células similares se han aislado en platelmintos parásitos como Echinococcus. Taenia solium causa la teniasis humana (intestinal) y la cisticercosis en humanos y cerdos. La infección del cerebro con larvas (quistes) de T. solium resulta en neurocisticercosis, hiperendémica en el Perú; su tratamiento se asocia a síntomas neurológicos graves. La capacidad proliferativa y el desarrollo de los estadios de T. solium aún no se describen, y no se ha caracterizado los neoblastos de este parásito. Se buscó células proliferativas en quistes de T. solium colectados de un cerdo infectado, que fueron identificadas al replicarse e incorporar el nucleótido bromodesoxiuridina, detectado con un anticuerpo monoclonal. Una línea celular estable de neoblastos sería útil para estudios sistemáticos in vitro sobre eficacia de drogas y sobre la biología de T. solium.

Neoblasts are totipotent cells, solely responsible for the proliferation and maturation of tissues in free-living flatworms. Similar cells have been isolated from parasitic flatworms such as Echinococcus. Taenia solium causes human taeniasis (intestinal) and cysticercosis in humans and pigs. Brain infection with larvae (cysts) of T. solium results in neurocysticercosis which is hyperendemic in Peru, and its treatment is associated with serious neurological symptoms. The proliferative capacity and development stages of T. solium have not been described and the neoblasts of this parasite have not been characterized We looked for cell proliferation in T. solium cysts collected from an infected pig, which were identified when replicating and incorporating bromodeoxyuridine nucleotide detected with a monoclonal antibody. A stable cell line of neoblasts would be useful for systematic in vitro studies on drug efficacy and the biology of T. solium.

Evans blue staining reveals vascular leakage associated with focal areas of host-parasite interaction in brains of pigs infected with Taenia solium.

Cysticidal drug treatment of viable Taenia solium brain parenchymal cysts leads to an acute pericystic host inflammatory response and blood brain barrier breakdown (BBB), commonly resulting in seizures. Naturally infected pigs, untreated or treated one time with praziquantel were sacrificed at 48 hr and 120 hr following the injection of Evans blue (EB) to assess the effect of treatment on larval parasites and surrounding tissue. Examination of harvested non encapsulated muscle cysts unexpectedly revealed one or more small, focal round region(s) of Evans blue dye infiltration (REBI) on the surface of otherwise non dye-stained muscle cysts. Histopathological analysis of REBI revealed focal areas of eosinophil-rich inflammatory infiltrates that migrated from the capsule into the tegument and internal structures of the parasite. In addition some encapsulated brain cysts, in which the presence of REBI could not be directly assessed, showed histopathology identical to that of the REBI. Muscle cysts with REBI were more frequent in pigs that had received praziquantel (6.6% of 3736 cysts; n = 6 pigs) than in those that were untreated (0.2% of 3172 cysts; n = 2 pigs). Similar results were found in the brain, where 20.7% of 29 cysts showed histopathology identical to muscle REBI cysts in praziquantel-treated pigs compared to the 4.3% of 47 cysts in untreated pigs. Closer examination of REBI infiltrates showed that EB was taken up only by eosinophils, a major component of the cellular infiltrates, which likely explains persistence of EB in the REBI. REBI likely represent early damaging host responses to T. solium cysts and highlight the focal nature of this initial host response and the importance of eosinophils at sites of host-parasite interaction. These findings suggest new avenues for immunomodulation to reduce inflammatory side effects of anthelmintic therapy.

[Identification of proliferating cells in Taenia solium cysts]. / Identificación de células proliferativas en quistes de Taenia solium.

Neoblasts are totipotent cells, solely responsible for the proliferation and maturation of tissues in free-living flatworms. Similar cells have been isolated from parasitic flatworms such as Echinococcus. Taenia solium causes human taeniasis (intestinal) and cysticercosis in humans and pigs. Brain infection with larvae (cysts) of T. solium results in neurocysticercosis which is hyperendemic in Peru, and its treatment is associated with serious neurological symptoms. The proliferative capacity and development stages of T. solium have not been described and the neoblasts of this parasite have not been characterized We looked for cell proliferation in T. solium cysts collected from an infected pig, which were identified when replicating and incorporating bromodeoxyuridine nucleotide detected with a monoclonal antibody. A stable cell line of neoblasts would be useful for systematic in vitro studies on drug efficacy and the biology of T. solium.

Disruption of the blood-brain barrier in pigs naturally infected with Taenia solium, untreated and after anthelmintic treatment.

Neurocysticercosis is a widely prevalent disease in the tropics that causes seizures and a variety into of neurological symptoms in most of the world. Experimental models are limited and do not allow assessment of the degree of inflammation around brain cysts. The vital dye Evans Blue (EB) was injected to 11 pigs naturally infected with Taenia solium cysts to visually identify the extent of disruption of the blood-brain barrier. A total of 369 cysts were recovered from the 11 brains and classified according to the staining of their capsules as blue or unstained. The proportion of cysts with blue capsules was significantly higher in brains from pigs that had received anthelmintic treatment 48 and 120h before the EB infusion, indicating a greater compromise of the blood-brain barrier due to treatment. The model could be useful for understanding the pathology of treatment-induced inflammation in neurocysticercosis.

In vitro analysis of albendazole sulfoxide enantiomers shows that (+)-(R)-albendazole sulfoxide is the active enantiomer against Taenia solium.

Albendazole is an anthelmintic drug widely used in the treatment of neurocysticercosis (NCC), an infection of the brain with Taenia solium cysts. However, drug levels of its active metabolite, albendazole sulfoxide (ABZSO), are erratic, likely resulting in decreased efficacy and suboptimal cure rates in NCC. Racemic albendazole sulfoxide is composed of ABZSO (+)-(R)- and (-)-(S) enantiomers that have been shown to differ in pharmacokinetics and activity against other helminths. The antiparasitic activities of racemic ABZSO and its (+)-(R)- and (-)-(S) enantiomers against T. solium cysts were evaluated in vitro. Parasites were collected from naturally infected pigs, cultured, and exposed to the racemic mixture or to each enantiomer (range, 10 to 500 ng/ml) or to praziquantel as a reference drug. The activity of each compound against cysts was assayed by measuring the ability to evaginate and inhibition of alkaline phosphatase (AP) and parasite antigen release. (+)-(R)-ABZSO was significantly more active than (-)-(S)-ABZSO in suppressing the release of AP and antigen into the supernatant in a dose- and time-dependent manner, indicating that most of the activity of ABZSO resides in the (+)-(R) enantiomer. Use of this enantiomer alone may lead to increased efficacy and/or less toxicity compared to albendazole.

Leishmania amazonensis META2 protein confers protection against heat shock and oxidative stress.

The META cluster of Leishmania amazonensis contains both META1 and META2 genes, which are upregulated in metacyclic promastigotes and encode proteins containing the META domain. Previous studies defined META2 as a 48.0-kDa protein, which is conserved in other Leishmania species and in Trypanosoma brucei. In this work, we demonstrate that META2 protein expression is regulated during the Leishmania life cycle but constitutive in T. brucei. META2 protein is present in the cytoplasm and flagellum of L. amazonensis promastigotes. Leishmania META2-null replacement mutants are more sensitive to oxidative stress and, upon heat shock, assume rounded morphology with shortened flagella. The increased susceptibility of null parasites to heat shock is reversed by extra-chromosomal expression of the META2 gene. Defective Leishmania promastigotes exhibit decreased ability to survive in macrophages. By contrast, META2 expression is decreased by 80% in RNAi-induced T. brucei bloodstream forms with no measurable effect on survival or resistance to heat shock.

Structural basis of molecular recognition of the Leishmania small hydrophilic endoplasmic reticulum-associated protein (SHERP) at membrane surfaces.

The 57-residue small hydrophilic endoplasmic reticulum-associated protein (SHERP) shows highly specific, stage-regulated expression in the non-replicative vector-transmitted stages of the kinetoplastid parasite, Leishmania major, the causative agent of human cutaneous leishmaniasis. Previous studies have demonstrated that SHERP localizes as a peripheral membrane protein on the cytosolic face of the endoplasmic reticulum and on outer mitochondrial membranes, whereas its high copy number suggests a critical function in vivo. However, the absence of defined domains or identifiable orthologues, together with lack of a clear phenotype in transgenic parasites lacking SHERP, has limited functional understanding of this protein. Here, we use a combination of biophysical and biochemical methods to demonstrate that SHERP can be induced to adopt a globular fold in the presence of anionic lipids or SDS. Cross-linking and binding studies suggest that SHERP has the potential to form a complex with the vacuolar type H(+)-ATPase. Taken together, these results suggest that SHERP may function in modulating cellular processes related to membrane organization and/or acidification during vector transmission of infective Leishmania.

TP0262 is a modulator of promoter activity of tpr Subfamily II genes of Treponema pallidum ssp. pallidum.

Transcriptional regulation in Treponema pallidum ssp. pallidum is poorly understood, primarily because this organism cannot be cultivated in vitro or genetically manipulated. We have recently shown a phase variation mechanism controlling transcription initiation of Subfamily II tpr (T. pallidumrepeat) genes (tprE, tprG and tprJ), a group of virulence factor candidates. Furthermore, the same study suggested that additional mechanisms might influence the level of transcription of these tprs. The T. pallidum genome sequence has revealed a few open reading frames with similarity to known bacterial transcription factors, including four catabolite activator protein homologues. In this work, sequences matching the Escherichia coli cAMP receptor protein (CRP) binding motif were identified in silico upstream of tprE, tprG and tprJ. Using elecrophoretic mobility shift assay and DNaseI footprinting assay, recombinant TP0262, a T. pallidum CRP homologue, was shown to bind specifically to amplicons obtained from the tpr promoters containing putative CRP binding motifs. Using a heterologous reporter system, binding of TP0262 to these promoters was shown to either increase (tprE and tprJ) or decrease (tprG) tpr promoter activity. This is the first characterization of a T. pallidum transcriptional modulator that influences tpr promoter activity.

Expression of the human RNA-binding protein HuR in Trypanosoma brucei increases the abundance of mRNAs containing AU-rich regulatory elements.

The salivarian trypanosome Trypanosoma brucei infects mammals and is transmitted by tsetse flies. The mammalian 'bloodstream form' trypanosome has a variant surface glycoprotein coat and relies on glycolysis while the procyclic form from tsetse flies has EP protein on the surface and has a more developed mitochondrion. We show here that the mRNA for the procyclic-specific cytosolic phosphoglycerate kinase PGKB, like that for EP proteins, contains a regulatory AU-rich element (ARE) that destabilises the mRNA in bloodstream forms. The human HuR protein binds to, and stabilises, mammalian mRNAs containing AREs. Expression of HuR in bloodstream-form trypanosomes resulted in growth arrest and in stabilisation of the EP, PGKB and pyruvate, phosphate dikinase mRNAs, while three bloodstream-specific mRNAs were reduced in abundance. The synthesis and abundance of unregulated mRNAs and proteins were unaffected. Our results suggest that regulation of mRNA stability by AREs arose early in eukaryotic evolution.

Compartmentation of enzymes in a microbody, the glycosome, is essential in Trypanosoma brucei.

All kinetoplastids contain membrane-bound microbodies known as glycosomes, in which several metabolic pathways including part of glycolysis are compartmentalized. Peroxin 2 is essential for the import of many proteins into the microbodies of yeasts and mammals. The PEX2 gene of Trypanosoma brucei was identified and its expression was silenced by means of tetracycline-inducible RNA interference. Bloodstream-form trypanosomes, which rely exclusively on glycolysis for ATP generation, died rapidly upon PEX2 depletion. Insect-form (procyclic) trypanosomes do not rely solely on glycolysis for ATP synthesis. PEX2 depletion in procyclic forms resulted in relocation of most tested matrix proteins to the cytosol, and these mutants also died. Compartmentation of microbody enzymes is therefore essential for survival of bloodstream and procyclic T. brucei. In contrast, yeasts and cultured mammalian cells grow normally in the absence of peroxisomal membranes unless placed on selective media.