Thiago M. Venancio

Schistosoma mansoni Tegument Protein Sm29 Is Able to Induce a Th1-Type of Immune Response and Protection against Parasite Infection

Background Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate. Methods and Findings We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p<0.05). Protective immunity in mice was associated with high titers of specific anti-Sm29 IgG1 and IgG2a and elevated production of IFN-γ, TNF-α and IL-12, a typical Th1 response. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to worms from control mice revealed a significant (q<0.01) down-regulation of 495 genes and up-regulation of only 22 genes. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals, suggesting that under immune attack schistosomes reduce the expression of critical surface proteins. Conclusion This study demonstrates that Sm29 surface protein is a new vaccine candidate against schistosomiasis and suggests that Sm29 vaccination associated with other protective critical surface antigens is the next logical strategy for improving protection.

Interference with Hemozoin Formation Represents an Important Mechanism of Schistosomicidal Action of Antimalarial Quinoline Methanols

Background The parasitic trematode Schistosoma mansoni is one of the major causative agents of human schistosomiasis, which afflicts 200 million people worldwide. Praziquantel remains the main drug used for schistosomiasis treatment, and reliance on the single therapy has been prompting the search for new therapeutic compounds against this disease. Our group has demonstrated that heme crystallization into hemozoin (Hz) within the S. mansoni gut is a major heme detoxification route with lipid droplets involved in this process and acting as a potential chemotherapeutical target. In the present work, we investigated the effects of three antimalarial compounds, quinine (QN), quinidine (QND) and quinacrine (QCR) in a murine schistosomiasis model by using a combination of biochemical, cell biology and molecular biology approaches. Methodology/Principal Findings Treatment of S. mansoni-infected female Swiss mice with daily intraperitoneal injections of QN, and QND (75 mg/kg/day) from the 11th to 17th day after infection caused significant decreases in worm burden (39%–61%) and egg production (42%–98%). Hz formation was significantly inhibited (40%–65%) in female worms recovered from QN- and QND-treated mice and correlated with reduction in the female worm burden. We also observed that QN treatment promoted remarkable ultrastructural changes in male and female worms, particularly in the gut epithelium and reduced the granulomatous reaction to parasite eggs trapped in the liver. Microarray gene expression analysis indicated that QN treatment increased the expression of transcripts related to musculature, protein synthesis and repair mechanisms. Conclusions The overall significant reduction in several disease burden parameters by the antimalarial quinoline methanols indicates that interference with Hz formation in S. mansoni represents an important mechanism of schistosomicidal action of these compounds and points out the heme crystallization process as a valid chemotherapeutic target to treat schistosomiasis.

The Aedes aegypti larval transcriptome: a comparative perspective with emphasis on trypsins and the domain structure of peritrophins

The genome sequence of Aedes aegypti was recently reported. A significant amount of Expressed Sequence Tags (ESTs) were sequenced to aid in the gene prediction process. In the present work we describe an integrated analysis of the genomic and EST data, focusing on genes with preferential expression in larvae (LG), adults (AG) and in both stages (SG). A total of 913 genes (5.4% of the transcript complement) are LG, including ion transporters and cuticle proteins that are important for ion homeostasis and defense. From a starting set of 245 genes encoding the trypsin domain, we identified 66 putative LG, AG, and SG trypsins by manual curation. Phylogenetic analyses showed that AG trypsins are divergent from their larval counterparts (LG), grouping with blood‐induced trypsins from Anopheles gambiae and Simulium vittatum. These results support the hypothesis that blood‐feeding arose only once, in the ancestral Culicomorpha. Peritrophins are proteins that interlock chitin fibrils to form the peritrophic membrane (PM) that compartmentalizes the food in the midgut. These proteins are recognized by having chitin‐binding domains with 6 conserved Cys and may also present mucin‐like domains (regions expected to be highly O‐glycosylated). PM may be formed by a ring of cells (type 2, seen in Ae. aegypti larvae and Drosophila melanogaster) or by most midgut cells (type 1, found in Ae. aegypti adult and Tribolium castaneum). LG and D. melanogaster peritrophins have more complex domain structures than AG and T. castaneum peritrophins. Furthermore, mucin‐like domains of peritrophins from T. castaneum (feeding on rough food) are lengthier than those of adult Ae. aegypti (blood‐feeding). This suggests, for the first time, that type 1 and type 2 PM may have variable molecular architectures determined by different peritrophins and/or ancillary proteins, which may be partly modulated by diet.

Reconstructing the ubiquitin network - cross-talk with other systems and identification of novel functions

BackgroundThe ubiquitin system (Ub-system) can be defined as the ensemble of components including Ub/ubiquitin-like proteins, their conjugation and deconjugation apparatus, binding partners and the proteasomal system. While several studies have concentrated on structure-function relationships and evolution of individual components of the Ub-system, a study of the system as a whole is largely lacking.ResultsUsing numerous genome-scale datasets, we assemble for the first time a comprehensive reconstruction of the budding yeast Ub-system, revealing static and dynamic properties. We devised two novel representations, the rank plot to understand the functional diversification of different components and the clique-specific point-wise mutual-information network to identify significant interactions in the Ub-system.ConclusionsUsing these representations, evidence is provided for the functional diversification of components such as SUMO-dependent Ub-ligases. We also identify novel components of SCF (Skp1-cullin-F-box)-dependent complexes, receptors in the ERAD (endoplasmic reticulum associated degradation) system and a key role for Sus1 in coordinating multiple Ub-related processes in chromatin dynamics. We present evidence for a major impact of the Ub-system on large parts of the proteome via its interaction with the transcription regulatory network. Furthermore, the dynamics of the Ub-network suggests that Ub and SUMO modifications might function cooperatively with transcription control in regulating cell-cycle-stage-specific complexes and in reinforcing periodicities in gene expression. Combined with evolutionary information, the structure of this network helps in understanding the lineage-specific expansion of SCF complexes with a potential role in pathogen response and the origin of the ERAD and ESCRT systems.

SmTRC1, a novel Schistosoma mansoni DNA transposon, discloses new families of animal and fungi transposons belonging to the CACTA superfamily

BackgroundThe CACTA (also called En/Spm) superfamily of DNA-only transposons contain the core sequence CACTA in their Terminal Inverted Repeats (TIRs) and so far have only been described in plants. Large transcriptome and genome sequence data have recently become publicly available for Schistosoma mansoni, a digenetic blood fluke that is a major causative agent of schistosomiasis in humans, and have provided a comprehensive repository for the discovery of novel genes and repetitive elements. Despite the extensive description of retroelements in S. mansoni, just a single DNA-only transposon belonging to the Merlin family has so far been reported in this organism.ResultsWe describe a novel S. mansoni transposon named SmTRC1, for S. mansoniT ransposon R elated to C ACTA 1, an element that shares several characteristics with plant CACTA transposons. Southern blotting indicates approximately 30–300 copies of SmTRC1 in the S. mansoni genome. Using genomic PCR followed by cloning and sequencing, we amplified and characterized a full-length and a truncated copy of this element. RT-PCR using S. mansoni mRNA followed by cloning and sequencing revealed several alternatively spliced transcripts of this transposon, resulting in distinct ORFs coding for different proteins. Interestingly, a survey of complete genomes from animals and fungi revealed several other novel TRC elements, indicating new families of DNA transposons belonging to the CACTA superfamily that have not previously been reported in these kingdoms. The first three bases in the S. mansoni TIR are CCC and they are identical to those in the TIRs of the insects Aedes aegypti and Tribolium castaneum, suggesting that animal TRCs may display a CCC core sequence.ConclusionThe DNA-only transposable element SmTRC1 from S. mansoni exhibits various characteristics, such as generation of multiple alternatively-spliced transcripts, the presence of terminal inverted repeats at the extremities of the elements flanked by direct repeats and the presence of a Transposase_21 domain, that suggest a distant relationship to CACTA transposons from Magnoliophyta. Several sequences from other Metazoa and Fungi code for proteins similar to those encoded by SmTRC1, suggesting that such elements have a common ancestry, and indicating inheritance through vertical transmission before separation of the Eumetazoa, Fungi and Plants.

Identification of the Schistosoma mansoni TNF-Alpha Receptor Gene and the Effect of Human TNF-Alpha on the Parasite Gene Expression Profile

Background Schistosoma mansoni is the major causative agent of schistosomiasis. The parasite takes advantage of host signals to complete its development in the human body. Tumor necrosis factor-alpha (TNF-α) is a human cytokine involved in skin inflammatory responses, and although its effect on the adult parasites metabolism and egg-laying process has been previously described, a comprehensive assessment of the TNF-α pathway and its downstream molecular effects is lacking. Methodology/Principal Findings In the present work we describe a possible TNF-α receptor (TNFR) homolog gene in S. mansoni (SmTNFR). SmTNFR encodes a complete receptor sequence composed of 599 amino acids, and contains four cysteine-rich domains as described for TNFR members. Real-time RT-PCR experiments revealed that SmTNFR highest expression level is in cercariae, 3.5 (±0.7) times higher than in adult worms. Downstream members of the known human TNF-α pathway were identified by an in silico analysis, revealing a possible TNF-α signaling pathway in the parasite. In order to simulate parasites exposure to human cytokine during penetration of the skin, schistosomula were exposed to human TNF-α just 3 h after cercariae-to-schistosomula in vitro transformation, and large-scale gene expression measurements were performed with microarrays. A total of 548 genes with significantly altered expression were detected, when compared to control parasites. In addition, treatment of adult worms with TNF-α caused a significantly altered expression of 1857 genes. Interestingly, the set of genes altered in adults is different from that of schistosomula, with 58 genes in common, representing 3% of altered genes in adults and 11% in 3 h-old early schistosomula. Conclusions/Significance We describe the possible molecular elements and targets involved in human TNF-α effect on S. mansoni, highlighting the mechanism by which recently transformed schistosomula may sense and respond to this host mediator at the site of cercarial penetration into the skin.

CYSTM, a novel cysteine-rich transmembrane module with a role in stress tolerance across eukaryotes

Using sensitive sequence profile analysis, we identify a hitherto uncharacterized cysteine-rich, transmembrane (TM) module, CYSTM, found in a wide range of tail-anchored membrane proteins across eukaryotes. This superfamily includes Schizosaccharomyces Uvi15, Arabidopsis PCC1, Digtaria CDT1 and Saccharomyces proteins YDL012C and YDR210W, which have all been implicated in resistance/response to stress or pathogens. Based on the pattern of conserved cysteines and data from different chemical genetics studies, we suggest that CYSTM proteins might have critical role in responding to deleterious compounds at the plasma membrane via chelation or redox-based mechanisms. Thus, CYSTM proteins are likely to be part of a novel cellular protective mechanism that is widely active in eukaryotes, including humans. Contact: aravind@ncbi.nih.gov Supplementary Information: Supplementary data are available at Bioinformatics online.

Effects of Phaseolus vulgaris (Fabaceae) seed coat on the embryonic and larval development of the cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae).

Bruchid beetles infest various seeds. The seed coat is the first protective barrier against bruchid infestation. Although non-host seed coats often impair the oviposition, eclosion and survival of the bruchid Callosobruchus maculatus larvae, morphological and biochemical aspects of this phenomenon remain unclear. Here we show that Phaseolus vulgaris (non-host) seed coat reduced C. maculatus female oviposition about 48%, increased 83% the seed penetration time, reduced larval mass and survival about 62 % and 40 % respectively. Interestingly, we found no visible effect on the major events of insect embryogenesis, namely the formation of the cellular blastoderm, germ band extension/retraction, embryo segmentation, appendage formation and dorsal closure. Larvae fed on P. vulgaris seed coat have greater FITC fluorescence signal in the midgut than in the feces, as opposed to what is observed in control larvae fed on Vigna unguiculata. Cysteine protease, α-amylase and α-glucosidase activities were reduced in larvae fed on P. vulgaris natural seed coat. Taken together, our results suggest that although P. vulgaris seed coat does not interfere with C. maculatus embryonic development, food digestion was clearly compromised, impacting larval fitness (e.g. body mass and survivability).

Reconstructing prokaryotic transcriptional regulatory networks: lessons from actinobacteria

Reconstruction of transcriptional regulatory networks of uncharacterized bacteria is a main challenge for the post-genomic era. Recent studies, including one in BMC Systems Biology, address this problem in the relatively underexplored actinobacteria clade, which includes major pathogenic and economically relevant taxa.

Heme Signaling Impacts Global Gene Expression, Immunity and Dengue Virus Infectivity in Aedes aegypti

Blood-feeding mosquitoes are exposed to high levels of heme, the product of hemoglobin degradation. Heme is a pro-oxidant that influences a variety of cellular processes. We performed a global analysis of heme-regulated Aedes aegypti (yellow fever mosquito) transcriptional changes to better understand influence on mosquito physiology at the molecular level. We observed an iron- and reactive oxygen species (ROS)-independent signaling induced by heme that comprised genes related to redox metabolism. By modulating the abundance of these transcripts, heme possibly acts as a danger signaling molecule. Furthermore, heme triggered critical changes in the expression of energy metabolism and immune response genes, altering the susceptibility towards bacteria and dengue virus. These findings seem to have implications on the adaptation of mosquitoes to hematophagy and consequently on their ability to transmit diseases. Altogether, these results may also contribute to the understanding of heme cell biology in eukaryotic cells.