Protein 2DE reference map of the anterior midgut of the blood-sucking bug Rhodnius prolixus.

Rhodnius prolixus is an important vector of Trypanosoma cruzi, the causative agent of Chagas' disease, an illness that affects 20% of Latin America population. The obligatory course of the parasite in the vector digestive tract has made it an important target for investigation in order to control the parasite transmission and thus interrupt its biological cycle in the insect vector. Therefore, an insight into the vector midgut physiology is valuable for insect control as well as to provide potential novel targets for drugs and vaccines development and thus disease treatment. In this study, the first 2DE map of R. prolixus anterior midgut is described. Proteins were separated by 2DE and analyzed by nano-LC MS/MS. The results yielded 489 proteins from 475 spots. These proteins were classified into 28 functional groups and their physiological roles in the insect midgut are discussed. All MS data have been deposited in the ProteomeXchange with identifiers PXD001488 and PXD001489 (http://proteomecentral.proteomexchange.org/dataset/PXD001488, http://proteomecentral.proteomexchange.org/dataset/PXD001489).

Trypanosoma brucei FKBP12 differentially controls motility and cytokinesis in procyclic and bloodstream forms.

FKBP12 proteins are able to inhibit TOR kinases or calcineurin phosphatases upon binding of rapamycin or FK506 drugs, respectively. The Trypanosoma brucei FKBP12 homologue (TbFKBP12) was found to be a cytoskeleton-associated protein with specific localization in the flagellar pocket area of the bloodstream form. In the insect procyclic form, RNA interference-mediated knockdown of TbFKBP12 affected motility. In bloodstream cells, depletion of TbFKBP12 affected cytokinesis and cytoskeleton architecture. These last effects were associated with the presence of internal translucent cavities limited by an inside-out configuration of the normal cell surface, with a luminal variant surface glycoprotein coat lined up by microtubules. These cavities, which recreated the streamlined shape of the normal trypanosome cytoskeleton, might represent unsuccessful attempts for cell abscission. We propose that TbFKBP12 differentially affects stage-specific processes through association with the cytoskeleton.

Cytokinesis of Trypanosoma brucei bloodstream forms depends on expression of adenylyl cyclases of the ESAG4 or ESAG4-like subfamily.

Antigenic variation of the parasite Trypanosoma brucei operates by monoallelic expression of a variant surface glycoprotein (VSG) from a collection of multiple telomeric expression sites (ESs). Each of these ESs harbours a long polycistronic transcription unit containing several expression site-associated genes (ESAGs). ESAG4 copies encode bloodstream stage-specific adenylyl cyclases (AC) and belong to a larger gene family of around 80 members, the majority of which, termed genes related to ESAG4 (GRESAG4s), are not encoded in ESs and are expressed constitutively in the life cycle. Here we report that ablation of ESAG4 from the active ES did not affect parasite growth, neither in culture nor upon rodent infection, and did not significantly change total AC activity. In contrast, inducible RNAi-mediated knock-down of an AC subfamily that includes ESAG4 and two ESAG4-like GRESAG4 (ESAG4L) genes, decreased total AC activity and induced a lethal phenotype linked to impaired cytokinesis. In the Δesag4 line compensatory upregulation of apparently functionally redundant ESAG4L genes was observed, suggesting that the ESAG4/ESAG4L-subfamily ACs are involved in the control of cell division. How deregulated adenylyl cyclases or cAMP might impair cytokinesis is discussed.

The GTPase TcRjl of the human pathogen Trypanosoma cruzi is involved in the cell growth and differentiation.

The protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas Disease, undergoes through a complex life cycle where rounds of cell division and differentiation occur initially in the gut of triatominae vectors and, after transmission, inside of infected cells in vertebrate hosts. Members of the Ras superfamily of GTPases are molecular switches which play pivotal regulatory functions in cell growth and differentiation. We have previously described a novel GTPase in T. cruzi, TcRjl, which belongs to the RJL family of Ras-related GTP binding proteins. Here we show that most of TcRjl protein is found bound to GTP nucleotides and may be locked in this stage. In addition, we show that TcRjl is located close to the kinetoplast, in a region corresponding possibly to flagellar pocket of the parasite and the expression of a dominant-negative TcRjl construct (TcRjlS37N) displays a significative growth phenotype in reduced serum medium. Remarkably, overexpression of TcRjl inhibits differentiation of epimastigotes to trypomastigote forms and promotes the accumulation of intermediate differentiation stages. Our data suggest that TcRjl might play a role in the control of the parasite growth and differentiation.

Rhodnius prolixus Hemolymph Immuno-Physiology: Deciphering the Systemic Immune Response Triggered by Trypanosoma cruzi Establishment in the Vector Using Quantitative Proteomics.

Understanding the development of Trypanosoma cruzi within the triatomine vector at the molecular level should provide novel targets for interrupting parasitic life cycle and affect vectorial competence. The aim of the current study is to provide new insights into triatomines immunology through the characterization of the hemolymph proteome of Rhodnius prolixus, a major Chagas disease vector, in order to gain an overview of its immune physiology. Surprisingly, proteomics investigation of the immunomodulation of T. cruzi-infected blood reveals that the parasite triggers an early systemic response in the hemolymph. The analysis of the expression profiles of hemolymph proteins from 6 h to 24 h allowed the identification of a broad range of immune proteins expressed already in the early hours post-blood-feeding regardless of the presence of the parasite, ready to mount a rapid response exemplified by the significant phenol oxidase activation. Nevertheless, we have also observed a remarkable induction of the immune response triggered by an rpPGRP-LC and the overexpression of defensins 6 h post-T. cruzi infection. Moreover, we have identified novel proteins with immune properties such as the putative c1q-like protein and the immunoglobulin I-set domain-containing protein, which have never been described in triatomines and could play a role in T. cruzi recognition. Twelve proteins with unknown function are modulated by the presence of T. cruzi in the hemolymph. Determining the function of these parasite-induced proteins represents an exciting challenge for increasing our knowledge about the diversity of the immune response from the universal one studied in holometabolous insects. This will provide us with clear answers for misunderstood mechanisms in host-parasite interaction, leading to the development of new generation strategies to control vector populations and pathogen transmission.

On the caveats of a multiplex test for SARS-CoV-2 to detect seroconversion after infection or vaccination.

The Covid-19 pandemic, caused by SARS-CoV-2, has resulted in over 6 million reported deaths worldwide being one of the biggest challenges the world faces today. Here we present optimizations of all steps of an enzyme-linked immunosorbent assay (ELISA)-based test to detect IgG, IgA and IgM against the trimeric spike (S) protein, receptor binding domain (RBD), and N terminal domain of the nucleocapsid (N-NTD) protein of SARS-CoV-2. We discuss how to determine specific thresholds for antibody positivity and its limitations according to the antigen used. We applied the assay to a cohort of 126 individuals from Rio de Janeiro, Brazil, consisting of 23 PCR-positive individuals and 103 individuals without a confirmed diagnosis for SARS-CoV-2 infection. To illustrate the differences in serological responses to vaccinal immunization, we applied the test in 18 individuals from our cohort before and after receiving ChAdOx-1 nCoV-19 or CoronaVac vaccines. Taken together, our results show that the test can be customized at different stages depending on its application, enabling the user to analyze different cohorts, saving time, reagents, or samples. It is also a valuable tool for elucidating the immunological consequences of new viral strains and monitoring vaccination coverage and duration of response to different immunization regimens.

IgA quantification as a good predictor of the neutralizing antibodies levels after vaccination against SARS-CoV-2.

Background: Vaccination against COVID-19 was implemented very quickly, but the emergence of new variants that can evade the previous acquired immunological protection highlights the importance of understanding the mechanisms involved in the immune response generated after SARS-CoV-2 infection or vaccination. Objectives: Since most of our knowledge on the humoral immunity generated against SARS-CoV-2 has been obtained from studies with infected patients before vaccination, our goal here was to evaluate seroconversion and its correlation with the titers of neutralizing antibodies (NAbs) in individuals who received the complete initial recommended vaccination schedule with three different vaccines. Study design: We analyzed serum IgG, IgA and total NAbs against the trimeric SARS-CoV-2 Spike (S) protein or its receptor binding domain (RBD) in blood samples collected from 118 healthy individuals without known previous infection, before and after receiving the first and the second dose of CoronaVac (n = 18), ChAdOx-1 (n = 68) or BNT162b2 (n = 32) vaccines. Results: We found that although IgG titers were high in all sera collected after the two doses of these vaccines, NAbs amounts varies among the groups. In contrast, serum NAbs concentrations were much more comparable to the IgA levels, indicating that these antibodies would have a major neutralizing capacity against SARS-CoV-2. Conclusions: Altogether our data suggest that quantification of serum anti-S or anti-RBD IgA, rather than IgG, may be a valuable tool to screen NAbs and may be considered for surveillance of vaccine coverage.

A multi-adenylate cyclase regulator at the flagellar tip controls African trypanosome transmission.

Signaling from ciliary microdomains controls developmental processes in metazoans. Trypanosome transmission requires development and migration in the tsetse vector alimentary tract. Flagellar cAMP signaling has been linked to parasite social motility (SoMo) in vitro, yet uncovering control of directed migration in fly organs is challenging. Here we show that the composition of an adenylate cyclase (AC) complex in the flagellar tip microdomain is essential for tsetse salivary gland (SG) colonization and SoMo. Cyclic AMP response protein 3 (CARP3) binds and regulates multiple AC isoforms. CARP3 tip localization depends on the cytoskeletal protein FLAM8. Re-localization of CARP3 away from the tip microdomain is sufficient to abolish SoMo and fly SG colonization. Since intrinsic development is normal in carp3 and flam8 knock-out parasites, AC complex-mediated tip signaling specifically controls parasite migration and thereby transmission. Participation of several developmentally regulated receptor-type AC isoforms may indicate the complexity of the in vivo signals perceived.

Early Post-Prandial Regulation of Protein Expression in the Midgut of Chagas Disease Vector Rhodnius prolixus Highlights New Potential Targets for Vector Control Strategy.

Chagas disease is a vector-borne parasitic disease caused by the flagellated protozoan Trypanosoma cruzi and transmitted to humans by a large group of bloodsucking triatomine bugs. Triatomine insects, such as Rhodnius prolixus, ingest a huge amount of blood in a single meal. Their midgut represents an important interface for triatomine-trypanosome interactions. Furthermore, the development of parasites and their vectorial transmission are closely linked to the blood feeding and digestion; thus, an understanding of their physiology is essential for the development of new strategies to control triatomines. In this study, we used label-free quantitative proteomics to identify and analyze the early effect of blood feeding on protein expression in the midgut of Rhodnius prolixus. We both identified and quantified 124 proteins in the anterior midgut (AM) and 40 in the posterior midgut (PM), which vary significantly 6 h after feeding. The detailed analysis of these proteins revealed their predominant involvement in the primary function of hematophagy, including proteases, proteases inhibitors, amino acids metabolism, primary metabolites processing, and protein folding. Interestingly, our proteomics data show a potential role of the AM in protein digestion. Moreover, proteins related to detoxification processes and innate immunity, which are largely accepted to be triggered by blood ingestion, were mildly modulated. Surprisingly, one third of blood-regulated proteins in the AM have unknown function. This work contributes to the improvement of knowledge on the digestive physiology of triatomines in the early hours post-feeding. It provides key information for selecting new putative targets for the development of triatomine control tools and their potential role in the vector competence, which could be applied to other vector species.

High-Throughput Identification of the Rhodnius prolixus Midgut Proteome Unravels a Sophisticated Hematophagic Machinery.

Chagas disease is one of the most common parasitic infections in Latin America, which is transmitted by hematophagous triatomine bugs, of which Rhodnius prolixus is the vector prototype for the study of this disease. The protozoan parasite Trypanosoma cruzi, the etiologic agent of this disease, is transmitted by the vector to humans through the bite wound or mucosa. The passage of the parasite through the digestive tract of its vector constitutes a key step in its developmental cycle. Herewith, by a using high-throughput proteomic tool in order to characterize the midgut proteome of R. prolixus, we describe a set of functional groups of proteins, as well as the biological processes in which they are involved. This is the first proteomic analysis showing an elaborated hematophagy machinery involved in the digestion of blood, among which, several families of proteases have been characterized. The evaluation of the activity of cathepsin D proteases in the anterior part of the digestive tract of the insect suggested the existence of a proteolytic activity within this compartment, suggesting that digestion occurs early in this compartment. Moreover, several heat shock proteins, blood clotting inhibitors, and a powerful antioxidant enzyme machinery against reactive oxygen species (ROS) and cell detoxification have been identified. Highlighting the complexity and importance of the digestive physiology of insects could be a starting point for the selection of new targets for innovative control strategies of Chagas disease.

Adenylate Cyclases of Trypanosoma brucei, Environmental Sensors and Controllers of Host Innate Immune Response.

Trypanosoma brucei, etiological agent of Sleeping Sickness in Africa, is the prototype of African trypanosomes, protozoan extracellular flagellate parasites transmitted by saliva (Salivaria). In these parasites the molecular controls of the cell cycle and environmental sensing are elaborate and concentrated at the flagellum. Genomic analyses suggest that these parasites appear to differ considerably from the host in signaling mechanisms, with the exception of receptor-type adenylate cyclases (AC) that are topologically similar to receptor-type guanylate cyclase (GC) of higher eukaryotes but control a new class of cAMP targets of unknown function, the cAMP response proteins (CARPs), rather than the classical protein kinase A cAMP effector (PKA). T. brucei possesses a large polymorphic family of ACs, mainly associated with the flagellar membrane, and these are involved in inhibition of the innate immune response of the host prior to the massive release of immunomodulatory factors at the first peak of parasitemia. Recent evidence suggests that in T. brucei several insect-specific AC isoforms are involved in social motility, whereas only a few AC isoforms are involved in cytokinesis control of bloodstream forms, attesting that a complex signaling pathway is required for environmental sensing. In this review, after a general update on cAMP signaling pathway and the multiple roles of cAMP, I summarize the existing knowledge of the mechanisms by which pathogenic microorganisms modulate cAMP levels to escape immune defense.

Potent immunosuppressive activity of a phosphodiesterase-4 inhibitor N-acylhydrazone in models of lipopolysaccharide-induced shock and delayed-type hypersensitivity reaction.

Immunosuppressive drugs are widely used for the treatment of immune-mediated diseases and inflammation, but the toxicity and side effects of the available immunosuppressors make the search of new agents of great relevance. Here, we evaluated the immunomodulatory activity of an N-acylhydrazone derivative, (E)-N'-(3,4-dimethoxybenzylidene)-4-methoxybenzohydrazide (LASSBio-1386), a phosphodiesterase-4 (PDE-4) inhibitor. LASSBio-1386 inhibited lymphocyte activation in a concentration-dependent fashion, decreasing lymphoproliferation and IFN-γ and IL-2 production stimulated by anti-CD3/CD28 mAbs or concanavalin A (Con A) and inducing cell-cycle arrest in the G0/G1 phase. These effects were not blocked by RU486, a glucocorticoid receptor (GR) antagonist, indicating an effect independent of glucocorticoid receptor activation. Combination index-isobologram analysis indicates a synergistic effect between LASSBio-1386 and dexamethasone in lymphoproliferation inhibition. LASSBio-1386 presented immunomodulatory action in macrophage cultures, as observed by a significant and concentration-dependent decrease in NO and TNF-α production, an effect achieved by reducing IĸB expression and NF-κB activation. In the mouse model of endotoxic shock, LASSBio-1386 at 50 and 100 mg/kg protected 50 and 85% of mice against LPS-induced lethality, respectively. In agreement to its in vitro action, treatment with 100 mg/kg of LASSBio-1386 reduced TNF-α and IL-1ß serum levels, while increased IL-6 and IL-10. Finally, LASSBio-1386 reduced the paw edema in a BSA-induced delayed-type hypersensitivity model. These findings demonstrate the immunomodulatory and immunosuppressant effects of LASSBio-1386 and indicate this molecule is a promising pharmacologic agent for immune-mediated diseases.

Solution structure and backbone dynamics of the Trypanosoma cruzi cysteine protease inhibitor chagasin.

A Trypanosoma cruzi cysteine protease inhibitor, termed chagasin, is the first characterized member of a new family of tight-binding cysteine protease inhibitors identified in several lower eukaryotes and prokaryotes but not present in mammals. In the protozoan parasite T.cruzi, chagasin plays a role in parasite differentiation and in mammalian host cell invasion, due to its ability to modulate the endogenous activity of cruzipain, a lysosomal-like cysteine protease. In the present work, we determined the solution structure of chagasin and studied its backbone dynamics by NMR techniques. Structured as a single immunoglobulin-like domain in solution, chagasin exerts its inhibitory activity on cruzipain through conserved residues placed in three loops in the same side of the structure. One of these three loops, L4, predicted to be of variable length among chagasin homologues, is flexible in solution as determined by measurements of (15)N relaxation. The biological implications of structural homology between chagasin and other members of the immunoglobulin super-family are discussed.

Identification and preliminary characterization of a putative C-type lectin receptor-like protein in the T. cruzi tomato lectin endocytic-enriched proteome.

Trypanosoma cruzi, the etiological agent of the Chagas' disease in Latin America undergoes a complex life cycle involving two hosts, a mammalian host and a reduviid insect vector (triatomine). In the insect midgut the parasite multiplies as epimastigote forms, which rely on endocytosis for their energy requirement. We recently showed that posttranslational modification of endocytic N-glycoproteins by tomato lectin (TL) binding-N-glycans is crucial for receptor-mediated endocytosis (RME) in epimastigote forms. In an attempt to characterize the endocytic proteome we used a TL affinity chromatography, which significantly enriched glycoproteins of the trypanosomal endocytic pathway. In addition to various lysosomal hydrolases, we found an endosomal C-type lectin-like protein, which displays some structural and topological characteristics of the mammalian lectin receptor superfamily. This lectin encoding a large transmembrane protein of around 375kDa contained three putative extracellular N-terminal C-type lectin domains (CTLD) and located inside the flagellar pocket (FP)/cytostome and endosomal compartments of the insect stage of the parasite and on the surface of the plasma membrane of intracellular amastigote parasites. Noteworthy, this endogenous lectin displayed similar sugar-binding specificity to that of TL and therefore could be important in either the N-glycan mediated endocytosis or parasite adhesion to host cells. We postulated that during the evolution of trypanosomatids, genes encoding lectin harboring 3 CTDLs represent an old acquisition present in free-living, monoxenic and heteroxenic trypanosomatids, which would have been secondarily lost in extracellular parasites from the T. brucei clade.

Specific Endocytosis Blockade of Trypanosoma cruzi Exposed to a Poly-LAcNAc Binding Lectin Suggests that Lectin-Sugar Interactions Participate to Receptor-Mediated Endocytosis.

Trypanosoma cruzi is a protozoan parasite transmitted by a triatomine insect, and causing human Chagas disease in South America. This parasite undergoes a complex life cycle alternating between non-proliferative and dividing forms. Owing to their high energy requirement, replicative epimastigotes of the insect midgut display high endocytic activity. This activity is mainly restricted to the cytostome, by which the cargo is taken up and sorted through the endosomal vesicular network to be delivered to reservosomes, the final lysosomal-like compartments. In African trypanosomes tomato lectin (TL) and ricin, respectively specific to poly-N-acetyllactosamine (poly-LacNAc) and ß-D-galactose, allowed the identification of giant chains of poly-LacNAc in N-glycoproteins of the endocytic pathway. We show that in T. cruzi epimastigote forms also, glycoproteins of the endocytic pathway are characterized by the presence of N-linked glycans binding to both ricin and TL. Affinity chromatography using both TL and Griffonia simplicifolia lectin II (GSLII), specific to non-reducing terminal residue of N-acetylglucosamine (GlcNAc), led to an enrichment of glycoproteins of the trypanosomal endocytic pathway. Incubation of live parasites with TL, which selectively bound to the cytostome/cytopharynx, specifically inhibited endocytosis of transferrin (Tf) but not dextran, a marker of fluid endocytosis. Taken together, our data suggest that N-glycan modification of endocytic components plays a crucial role in receptor-mediated endocytosis of T. cruzi.

Simultaneous depletion of Atm and Mdl rebalances cytosolic Fe-S cluster assembly but not heme import into the mitochondrion of Trypanosoma brucei.

ABC transporter mitochondrial 1 (Atm1) and multidrug resistance-like 1 (Mdl) are mitochondrial ABC transporters. Although Atm1 was recently suggested to transport different forms of glutathione from the mitochondrion, which are used for iron-sulfur (Fe-S) cluster maturation in the cytosol, the function of Mdl remains elusive. In Trypanosoma brucei, we identified one homolog of each of these genes, TbAtm and TbMdl, which were downregulated either separately or simultaneously using RNA interference. Individual depletion of TbAtm and TbMdl led to limited growth defects. In cells downregulated for TbAtm, the enzymatic activities of the Fe-S cluster proteins aconitase and fumarase significantly decreased in the cytosol but not in the mitochondrion. Downregulation of TbMdl did not cause any change in activities of the Fe-S proteins. Unexpectedly, the simultaneous downregulation of TbAtm and TbMdl did not result in any growth defect, nor were the Fe-S cluster protein activities altered in either the cytosolic or mitochondrial compartments. Additionally, TbAtm and TbMdl were able to partially restore the growth of the Saccharomyces cerevisiae Δatm1 and Δmdl2 null mutants, respectively. Because T. brucei completely lost the heme b biosynthesis pathway, this cofactor has to be obtained from the host. Based on our results, TbMdl is a candidate for mitochondrial import of heme b, which was markedly decreased in both TbMdl and TbAtm + TbMdl knockdowns. Moreover, the levels of heme a were strongly decreased in the same knockdowns, suggesting that TbMdl plays a key role in heme a biosynthesis, thus affecting the overall heme homeostasis in T. brucei.

In vitro metacyclogenesis of Trypanosoma cruzi induced by starvation correlates with a transient adenylyl cyclase stimulation as well as with a constitutive upregulation of adenylyl cyclase expression.

The Trypanosoma cruzi adenylyl cyclase (AC) multigene family encodes different isoforms (around 15) sharing a variable large N-terminal domain, which is extracellular and receptor-like, followed by a transmembrane helix and a conserved C-terminal catalytic domain. It was proposed that these key enzymes in the cAMP signalling pathway allow the parasite to sense its changing extracellular milieu in order to rapidly adapt to its new environment, which is generally achieved through a differentiation process. One of the critical differentiation events the parasitic protozoan T. cruzi undergoes during its life cycle, known as metacyclogenesis, occurs in the digestive tract of the insect and corresponds to the differentiation from noninfective epimastigotes to infective metacyclic trypomastigote forms. By in vitro monitoring the activity of AC during metacyclogenesis, we showed that both the activity of AC and the intracellular cAMP content follow a similar pattern of transient stimulation in a two-step process, with a first activation peak occurring during the first hours of nutritional stress and a second peak between 6 and 48 h, corresponding to the cellular adhesion. During this differentiation process, a general mechanism of upregulation of AC expression of both mRNA and protein is triggered and in particular for a major subclass of these enzymes that are present in various gene copies commonly associated to the THT gene clusters. Although the scattered genome distribution of these gene copies is rather unusual in trypanosomatids and seems to be a recent acquisition in the evolution of the T. cruzi clade, their encoded product redistributed on the flagellum of the parasite upon differentiation could be important to sense the extracellular milieu.

Monitoring of the Parasite Load in the Digestive Tract of Rhodnius prolixus by Combined qPCR Analysis and Imaging Techniques Provides New Insights into the Trypanosome Life Cycle.

BACKGROUND: Here we report the monitoring of the digestive tract colonization of Rhodnius prolixus by Trypanosoma cruzi using an accurate determination of the parasite load by qPCR coupled with fluorescence and bioluminescence imaging (BLI). These complementary methods revealed critical steps necessary for the parasite population to colonize the insect gut and establish vector infection. METHODOLOGY/PRINCIPAL FINDINGS: qPCR analysis of the parasite load in the insect gut showed several limitations due mainly to the presence of digestive-derived products that are thought to degrade DNA and inhibit further the PCR reaction. We developed a real-time PCR strategy targeting the T. cruzi repetitive satellite DNA sequence using as internal standard for normalization, an exogenous heterologous DNA spiked into insect samples extract, to precisely quantify the parasite load in each segment of the insect gut (anterior midgut, AM, posterior midgut, PM, and hindgut, H). Using combined fluorescence microscopy and BLI imaging as well as qPCR analysis, we showed that during their journey through the insect digestive tract, most of the parasites are lysed in the AM during the first 24 hours independently of the gut microbiota. During this short period, live parasites move through the PM to establish the onset of infection. At days 3-4 post-infection (p.i.), the parasite population begins to colonize the H to reach a climax at day 7 p.i., which is maintained during the next two weeks. Remarkably, the fluctuation of the parasite number in H remains relatively stable over the two weeks after refeeding, while the populations residing in the AM and PM increases slightly and probably constitutes the reservoirs of dividing epimastigotes. CONCLUSIONS/SIGNIFICANCE: These data show that a tuned dynamic control of the population operates in the insect gut to maintain an equilibrium between non-dividing infective trypomastigote forms and dividing epimastigote forms of the parasite, which is crucial for vector competence.

¹H, ¹5N and ¹³C assignments of a putative peptidyl prolyl cis-trans isomerase FKBP12 from Trypanosoma brucei.

TbFKBP12 is a putative peptidyl prolyl cis-trans isomerase from Trypanosoma brucei, causative agent of the African trypanosomiasis or sleeping sickness. It interacts with the immunosuppressive drug rapamycin inhibiting the formation of TORC2 complex leading to parasite death by inhibiting cell proliferation through cytokinesis blockade. Moreover, RNAi silencing of TbFKBP12 revealed essential function in both procyclic and bloodstream forms. Both facts make TbFKBP12 an attractive target for ligand development and thus structural data is desirable. In this work we report the NMR resonance assignments for (1)H, (15)N and (13)C nuclei in the backbone and side chains of the TbFKBP12 as basis for further studies of structure, backbone dynamics, interaction mapping and drug screening.