Influence of CK2 protein kinase activity on the interaction between Trypanosoma cruzi and its vertebrate and invertebrate hosts.

Chagas disease, endemic from Latin America, is caused by Trypanosoma cruzi and is transmitted by triatomine feces. This parasite undergoes complex morphological changes through its life cycle, promoted by significant changes in signal transduction pathways. The activity of protein kinase CK2 has been described in trypanosomatids. Using a specific peptide and radioactive ATP, we identified CK2 activity on the cellular surface and the cytoplasmic content in Trypanosoma cruzi, apart from the secreted form. Dephosphorylated casein promoted an increase of 48% in the secreted CK2 activity. Total extract of peritoneal macrophages from BALB/c and inactivated human serum promoted an increase of 67% and 36%, respectively, in this activity. The protein secreted by parasites was purified by HPLC and had shown compatibility with the catalytic subunit of mammalian CK2. Incubation of the parasites with CK2 inhibitors, added to the culture medium, prevented their growth. The opposite was observed when CK2 activators were used. Results of interaction between Trypanosoma cruzi and the gut of the vector have revealed that, in the presence of CK2 inhibitors, there is a reduction in the association rate. A similar inhibition profile was seen in the Trypanosoma cruzi-macrophages interaction, confirming the importance of this enzyme in the life cycle of this protozoan.

Effect of Larval Food Availability on Adult Aedes Aegypti (Diptera: Culicidae) Fitness and Susceptibility to Zika Infection.

Aedes (Stegomyia) aegypti (Linnaeus, 1762) is a mosquito species of significant medical importance. The use of this vector in research studies usually requires a large number of mosquitoes as well as rearing and maintenance in a laboratory-controlled environment. However, laboratory conditions may be different from field environments, presenting stressful challenges such as low food concentration, especially during larval stages, which may, in turn, impair vector biology. Therefore, we tested herein if larval food availability (0.004, 0.009, 0.020, and 0.070% diets) would affect overall adult insect fitness. We observed slower development in mosquitoes fed a 0.004% diet 15 d post-eclosion (DPE) and shorter mean time in mosquitoes fed a 0.020% diet (7 DPE). Larval diet and adult mosquito weight were positively correlated, and heavier females fed higher larval diets exhibited greater blood feeding capacity and oviposition. In addition, larval diet concentrations led to median adult lifespan variations (male/female in days-0.004%: 30 ± 1.41, 45 ± 1.3; 0.009%: 31.5 ± 1.33, 41 ± 1.43; 0.020%: 26 ± 1.18, 41 ± 1.45; 0.070%: 29 ± 1.07, 44 ± 1.34), reduced tolerance to deltamethrin (1 mg/m2) and changes in detoxification enzyme activities. Moreover, in the larval 0.070% diet, females presented higher Zika susceptibility (plaque-forming unit [PFU]: 1.218 × 106) compared with other diets (0.004%: 1.31 × 105; 0.009%: 2.0 × 105; 0.020%: 1.25 × 105 PFU). Altogether, our study demonstrates that larval diet restriction results not only in larval developmental arrest but also in adult fitness impairment, which must be considered in future assessments.

Insecticide resistance, fitness and susceptibility to Zika infection of an interbred Aedes aegypti population from Rio de Janeiro, Brazil.

BACKGROUND: Aedes aegypti is a vector of high relevance, since it transmits several arboviruses, including dengue, chikungunya and Zika. Studies on vector biology are usually conducted with laboratory strains presenting a divergent genetic composition from field populations. This may impair vector control policies that were based on laboratory observations employing only long maintained laboratory strains. In the present study we characterized a laboratory strain interbreed with Ae. aegypti collected from five different localities in Rio de Janeiro (Aedes Rio), for insecticide resistance (IR), IR mechanisms, fitness and Zika virus infection. METHODS: We compared the recently established Aedes Rio with the laboratory reference strain Rockefeller. Insecticide resistance (deltamethrin, malathion and temephos), activity of metabolic resistance enzymes and kdr mutation frequency were determined. Some life table parameters (longevity, blood-feeding, number and egg viability) and Zika virus susceptibility was also determined. RESULTS: Aedes Rio showed resistance to deltamethrin (resistance ratio, RR50 = 32.6) and temephos (RR50 = 7.0) and elevated activity of glutathione S-transferase (GST) and esterases (α-EST and pNPA-EST), but not acetylcholinesterase (AChE). In total, 92.1% of males genotyped for kdr presented a "resistant" genotype. Weekly blood-fed females from both strains, presented reduced mortality compared to sucrose-fed mosquitoes; however, Aedes Rio blood-fed females did not live as long (mean lifespan: Rockefeller = 70 ± 3.07; Aedes Rio = 53.5 ± 2.16 days). There were no differences between strains in relation to blood-feeding and number of eggs, but Aedes Rio eggs presented reduced viability (mean hatch: Rockefeller = 77.79 ± 1.4%; Aedes Rio = 58.57 ± 1.77%). Zika virus infection (plaque-forming unit, PFU) was similar in both strains (mean PFU ± SE: Aedes Rio: 4.53 × 104 ± 1.14 × 104 PFU; Rockefeller: 2.02 × 104 ± 0.71 × 104 PFU). CONCLUSION: Selected conditions in the field, such as IR mechanisms, may result in pleiotropic effects that interfere in general physiology of the insect. Therefore, it is important to well characterize field populations to be tested in parallel with laboratory reference strains. This practice would improve the significance of laboratory tests for vector control methods.

Bacterial community composition in the salivary glands of triatomines (Hemiptera: Reduviidae).

BACKGROUND: Chagas disease is caused by the parasite Trypanosoma cruzi and is transmitted through triatomines (Hemiptera: Reduviidae). In the last year, many studies of triatomine gut microbiota have outlined its potential role in modulating vector competence. However, little is known about the microbiota present in the salivary glands of triatomines. Bacterial composition of salivary glands in selected triatomine species was investigated, as well as environmental influences on the acquisition of bacterial communities. METHODOLOGY/PRINCIPAL FINDINGS: The diversity of the bacterial communities of 30 pairs of salivary glands of triatomines was studied by sequencing of the V1- V3 variable region of the 16S rRNA using the MiSeq platform (Illumina), and bacteria isolated from skin of three vertebrate hosts were identified based on 16S rRNA gene sequence analysis (targeting the V3-V5 region). In a comparative analysis of microbiota in the salivary glands of triatomine species, operational taxonomic units belonging to Arsenophonous appeared as dominant in Triatoma spp (74% of the total 16S coverage), while these units belonging to unclassified Enterobacteriaceae were dominant in the Rhodnius spp (57% of the total 16S coverage). Some intraspecific changes in the composition of the triatomine microbiota were observed, suggesting that some bacteria may have been acquired from the environment. CONCLUSIONS AND SIGNIFICANCE: Our study revealed the presence of a low-diversity microbiota associated to the salivary glands of the evaluated triatomines. The predominant bacteria genera are associated with triatomine genera and the bacteria can be acquired in the environment in which the insects reside. Further studies are necessary to determine the influence of bacterial communities on vector competence.

Molecular and functional characterization of single-box high-mobility group B (HMGB) chromosomal protein from Aedes aegypti.

High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein AaHMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, AaHMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for AaHMGB and AaHMGB1 during embryogenesis. Immunolocalization in the midgut revealed that AaHMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that AaHMGB exhibits common features of α-helical structures and is more stably folded than AaHMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between AaHMGB and AaHMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of AaHMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis.

Triatoma infestans relies on salivary lysophosphatidylcholine to enhance Trypanosoma cruzi transmission.

Triatoma infestans is a mandatory haematophagous vector of Chagas disease in Brazil. Despite a large number of studies on the anti-haemostatic molecules present in its saliva, the role of its salivary components on parasite transmission is poorly understood. Here, we show that the bioactive lipid molecule, lysophosphatidylcholine (LPC), is present in the salivary gland of T. infestans. We characterized the lipid profiles of each unit of the T. infestans salivary gland. We noticed that LPC is present in the three units of the salivary gland and that the insect feeding state does not influence its proportion. T. infestans saliva and LPC can enhance T. cruzi transmission to mice by dramatically altering the profile of inflammatory cells at the site of inoculation on mouse skin, facilitating the transmission of T. cruzi to the vertebrate host. Consequently, the mortality curves of either saliva- or LPC-injected mice display significant higher mortality rates than the control. Altogether, these results implicate LPC as one of key salivary molecule involved in Chagas disease transmission.

Polyphenol-Rich Diets Exacerbate AMPK-Mediated Autophagy, Decreasing Proliferation of Mosquito Midgut Microbiota, and Extending Vector Lifespan.

BACKGROUND: Mosquitoes feed on plant-derived fluids such as nectar and sap and are exposed to bioactive molecules found in this dietary source. However, the role of such molecules on mosquito vectorial capacity is unknown. Weather has been recognized as a major determinant of the spread of dengue, and plants under abiotic stress increase their production of polyphenols. RESULTS: Here, we show that including polyphenols in mosquito meals promoted the activation of AMP-dependent protein kinase (AMPK). AMPK positively regulated midgut autophagy leading to a decrease in bacterial proliferation and an increase in vector lifespan. Suppression of AMPK activity resulted in a 6-fold increase in midgut microbiota. Similarly, inhibition of polyphenol-induced autophagy induced an 8-fold increase in bacterial proliferation. Mosquitoes maintained on the polyphenol diet were readily infected by dengue virus. CONCLUSION: The present findings uncover a new direct route by which exacerbation of autophagy through activation of the AMPK pathway leads to a more efficient control of mosquito midgut microbiota and increases the average mosquito lifespan. Our results suggest for the first time that the polyphenol content and availability of the surrounding vegetation may increase the population of mosquitoes prone to infection with arboviruses.

CK2 Secreted by Leishmania braziliensis Mediates Macrophage Association Invasion: A Comparative Study between Virulent and Avirulent Promastigotes.

CK2 is a protein kinase distributed in different compartments of Leishmania braziliensis: an externally oriented ecto-CK2, an intracellular CK2, and a secreted CK2. This latter form is constitutively secreted from the parasite (CsCK2), but such secretion may be highly enhanced by the association of specific molecules, including enzyme substrates, which lead to a higher enzymatic activity, called inductively secreted CK2 (IsCK2). Here, we examined the influence of secreted CK2 (sCK2) activity on the infectivity of a virulent L. braziliensis strain. The virulent strain presented 121-fold higher total CK2 activity than those found in an avirulent strain. The use of specific CK2 inhibitors (TBB, DRB, or heparin) inhibited virulent parasite growth, whereas no effect was observed in the avirulent parasites. When these inhibitors were added to the interaction assays between the virulent L. braziliensis strain and macrophages, association index was drastically inhibited. Polyamines enhanced sCK2 activity and increased the association index between parasites and macrophages. Finally, sCK2 and the supernatant of the virulent strain increased the association index between the avirulent strain and macrophages, which was inhibited by TBB. Thus, the kinase enzyme CK2 seems to be important to invasion mechanisms of L. braziliensis.

Molecular analysis of Aedes aegypti classical protein tyrosine phosphatases uncovers an ortholog of mammalian PTP-1B implicated in the control of egg production in mosquitoes.

BACKGROUND: Protein Tyrosine Phosphatases (PTPs) are enzymes that catalyze phosphotyrosine dephosphorylation and modulate cell differentiation, growth and metabolism. In mammals, PTPs play a key role in the modulation of canonical pathways involved in metabolism and immunity. PTP1B is the prototype member of classical PTPs and a major target for treating human diseases, such as cancer, obesity and diabetes. These signaling enzymes are, hence, targets of a wide array of inhibitors. Anautogenous mosquitoes rely on blood meals to lay eggs and are vectors of the most prevalent human diseases. Identifying the mosquito ortholog of PTP1B and determining its involvement in egg production is, therefore, important in the search for a novel and crucial target for vector control. METHODOLOGY/PRINCIPAL FINDINGS: We conducted an analysis to identify the ortholog of mammalian PTP1B in the Aedes aegypti genome. We identified eight genes coding for classical PTPs. In silico structural and functional analyses of proteins coded by such genes revealed that four of these code for catalytically active enzymes. Among the four genes coding for active PTPs, AAEL001919 exhibits the greatest degree of homology with the mammalian PTP1B. Next, we evaluated the role of this enzyme in egg formation. Blood feeding largely affects AAEL001919 expression, especially in the fat body and ovaries. These tissues are critically involved in the synthesis and storage of vitellogenin, the major yolk protein. Including the classical PTP inhibitor sodium orthovanadate or the PTP substrate DiFMUP in the blood meal decreased vitellogenin synthesis and egg production. Similarly, silencing AAEL001919 using RNA interference (RNAi) assays resulted in 30% suppression of egg production. CONCLUSIONS/SIGNIFICANCE: The data reported herein implicate, for the first time, a gene that codes for a classical PTP in mosquito egg formation. These findings raise the possibility that this class of enzymes may be used as novel targets to block egg formation in mosquitoes.

Triatominae biochemistry goes to school: evaluation of a novel tool for teaching basic biochemical concepts of Chagas disease vectors.

We evaluate a new approach to teaching the basic biochemistry mechanisms that regulate the biology of Triatominae, major vectors of Trypanosoma cruzi, the causative agent of Chagas disease. We have designed and used a comic book, "Carlos Chagas: 100 years after a hero's discovery" containing scientific information obtained by seven distinguished contemporary Brazilian researchers working with Triatominaes. Students (22) in the seventh grade of a public elementary school received the comic book. The study was then followed up by the use of Concept Maps elaborated by the students. Six Concept Maps elaborated by the students before the introduction of the comic book received an average score of 7. Scores rose to an average of 45 after the introduction of the comic book. This result suggests that a more attractive content can greatly improve the knowledge and conceptual understanding among students not previously exposed to insect biochemistry. In conclusion, this study illustrates an alternative to current strategies of teaching about the transmission of neglected diseases. It also promotes the diffusion of the scientific knowledge produced by Brazilian researchers that may stimulate students to choose a scientific career.

Lysophosphatidylcholine triggers TLR2- and TLR4-mediated signaling pathways but counteracts LPS-induced NO synthesis in peritoneal macrophages by inhibiting NF-κB translocation and MAPK/ERK phosphorylation.

BACKGROUND: Lysophosphatidylcholine (LPC) is the main phospholipid component of oxidized low-density lipoprotein (oxLDL) and is usually noted as a marker of several human diseases, such as atherosclerosis, cancer and diabetes. Some studies suggest that oxLDL modulates Toll-like receptor (TLR) signaling. However, effector molecules that are present in oxLDL particles and can trigger TLR signaling are not yet clear. LPC was previously described as an attenuator of sepsis and as an immune suppressor. In the present study, we have evaluated the role of LPC as a dual modulator of the TLR-mediated signaling pathway. METHODOLOGY/PRINCIPAL FINDINGS: HEK 293A cells were transfected with TLR expression constructs and stimulated with LPC molecules with different fatty acid chain lengths and saturation levels. All LPC molecules activated both TLR4 and TLR2-1 signaling, as evaluated by NF-қB activation and IL-8 production. These data were confirmed by Western blot analysis of NF-қB translocation in isolated nuclei of peritoneal murine macrophages. However, LPC counteracted the TLR4 signaling induced by LPS. In this case, NF-қB translocation, nitric oxide (NO) synthesis and the expression of inducible nitric oxide synthase (iNOS) were blocked. Moreover, LPC activated the MAP Kinases p38 and JNK, but not ERK, in murine macrophages. Interestingly, LPC blocked LPS-induced ERK activation in peritoneal macrophages but not in TLR-transfected cells. CONCLUSIONS/SIGNIFICANCE: The above results indicate that LPC is a dual-activity ligand molecule. It is able to trigger a classical proinflammatory phenotype by activating TLR4- and TLR2-1-mediated signaling. However, in the presence of classical TLR ligands, LPC counteracts some of the TLR-mediated intracellular responses, ultimately inducing an anti-inflammatory phenotype; LPC may thus play a role in the regulation of cell immune responses and disease progression.

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus.

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

Glycoinositolphospholipids from Trypanosomatids subvert nitric oxide production in Rhodnius prolixus salivary glands.

BACKGROUND: Rhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands. METHODOLOGY/PRINCIPAL FINDINGS: Here we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities. CONCLUSIONS/SIGNIFICANCE: Taken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.

The dengue vector Aedes aegypti contains a functional high mobility group box 1 (HMGB1) protein with a unique regulatory C-terminus.

The mosquito Aedes aegypti can spread the dengue, chikungunya and yellow fever viruses. Thus, the search for key molecules involved in the mosquito survival represents today a promising vector control strategy. High Mobility Group Box (HMGB) proteins are essential nuclear factors that maintain the high-order structure of chromatin, keeping eukaryotic cells viable. Outside the nucleus, secreted HMGB proteins could alert the innate immune system to foreign antigens and trigger the initiation of host defenses. In this work, we cloned and functionally characterized the HMGB1 protein from Aedes aegypti (AaHMGB1). The AaHMGB1 protein typically consists of two HMG-box DNA binding domains and an acidic C-terminus. Interestingly, AaHMGB1 contains a unique alanine/glutamine-rich (AQ-rich) C-terminal region that seems to be exclusive of dipteran HMGB proteins. AaHMGB1 is localized to the cell nucleus, mainly associated with heterochromatin. Circular dichroism analyses of AaHMGB1 or the C-terminal truncated proteins revealed α-helical structures. We showed that AaHMGB1 can effectively bind and change the topology of DNA, and that the AQ-rich and the C-terminal acidic regions can modulate its ability to promote DNA supercoiling, as well as its preference to bind supercoiled DNA. AaHMGB1 is phosphorylated by PKA and PKC, but not by CK2. Importantly, phosphorylation of AaHMGB1 by PKA or PKC completely abolishes its DNA bending activity. Thus, our study shows that a functional HMGB1 protein occurs in Aedes aegypt and we provide the first description of a HMGB1 protein containing an AQ-rich regulatory C-terminus.

CK2 phosphorylation of Schistosoma mansoni HMGB1 protein regulates its cellular traffic and secretion but not its DNA transactions.

BACKGROUND: The helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. PRINCIPAL FINDINGS: We showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated. CONCLUSIONS: We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis.

Functional properties of Schistosoma mansoni single-stranded DNA-binding protein SmPUR-alpha. Description of the interaction between SmPUR-alpha and SMYB1.

PUR-alpha is a highly conserved protein in eukaryotes belonging to the family of single-stranded DNA-binding proteins. Because PUR-alpha is a multifunctional protein that participates in several regulatory events at the level of gene transcription, it became relevant to investigate the structural features of Schistosoma mansoni PUR-alpha (SmPUR-alpha) that could be correlated to its mode of action. Using deletion constructs on a dot blot assay we mapped the domains of GST-SmPUR-alpha fusion protein involved in the interactions with DNA and RNA. Individually, the N-terminal amino acid residues 1-26 and the C-terminal residues 196-276 of GST-SmPUR-alpha which did not contain nucleic acid-binding domains, did not bind ssDNA or RNA. In contrast, domains encompassing the N-terminal and Class I and C-terminal plus Class I exhibited the highest binding affinity. Seemingly, the latter (GST-SmPUR-alpha 174-276) played a major role in nucleic acid interaction as judged by affinity alone. Other combinations of the deletion constructs displayed either intermediary or no binding affinity to the DNA or RNA probes. Gel shift competition assay showed that GST-SmPUR-alpha bound to ssDNA with higher affinity than to RNA. Because SmPUR-alpha contains two putative phosphorylation sites the protein was tested as a substrate to casein kinase II. GST-SmPUR-alpha could be phosphorylated in vitro by casein kinase II at both, the N- and C-terminus of the protein. The multifunctional nature of SmPUR-alpha was demonstrated by experiments measuring the physical interaction between SmPUR-alpha and the transcription factor SMYB1. This was determined in vivo (yeast two hybrid) and in vitro (GST-pull down). Furthermore, we showed that SmPUR-alpha and SMYB1 acted synergistically to bind preferentially to pyrimidine-rich sequences.