Potential Correlation between Carboxylic Acid Metabolites in Biomphalaria alexandrina Snails after Exposure to Schistosoma mansoni Infection

Carboxylic acids play an important role in both aerobic and anaerobic metabolic pathways of both the snail and the parasite. Monitoring the effects of infection by schistosome on Biomphalaria alexandrina carboxylic acids metabolic profiles represents a promising additional source of information about the state of metabolic system. We separated and quantified pyruvic, fumaric, malic, oxalic, and acetic acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to detect correlations between these acids in both hemolymph and digestive gland gonad complex (DGG's) samples in a total of 300 B. alexandrina snails (150 infected and 150 controls) at different stages of infection. The results showed that the majority of metabolite pairs did not show significant correlations. However, some high correlations were found between the studied acids within the control group but not in other groups. More striking was the existence of reversed correlations between the same acids at different stages of infection. Some possible explanations of the underlying mechanisms were discussed. Ultimately, however, further data are required for resolving the responsible regulatory events. These findings highlight the potential of metabolomics as a novel approach for fundamental investigations of host-pathogen interactions as well as disease surveillance and control.

Proteomic analysis of the shistosome tegument and its surface membranes

The tegument surface of the adult schistosome, bounded by a normal plasma membrane overlain by a secreted membranocalyx, holds the key to understanding how schistosomes evade host immune responses. Recent advances in mass spectrometry (MS), and the sequencing of the Schistosoma mansoni transcriptome/genome, have facilitated schistosome proteomics. We detached the tegument from the worm body and enriched its surface membranes by differential extraction, before subjecting the preparation to liquid chromatography-based proteomics to identify its constituents. The most exposed proteins on live worms were labelled with impearmeant biotinylation reagents, and we also developed methods to isolate the membranocalyx for analysis. We identified transporters for sugars, amino acids, inorganic ions and water, which confirm the importance of the tegument plasma membrane in nutrient acquisition and solute balance. Enzymes, including phosphohydrolases, esterases and carbonic anhydrase were located with their catalytic domains external to the plasma membrane, while five tetraspanins, annexin and dysferlin were implicated in membrane architecture. In contrast, few parasite proteins could be assigned to the membranocalyx but mouse immune response proteins, including three immunoglobulins and two complement factors, were detected, plus host membrane proteins such as CD44, integrin and a complement regulatory protein, testifying to the acquisitive properties of the secreted bilayer.

Identification of a new Schistosoma mansoni membrane-bound protein through bioinformatic analysis

Progress in schistosome genome research has enabled investigators to move rapidly from genome sequences to vaccine development. Proteins bound to the surface of parasites are potential vaccine candidates, or they can be used for diagnosis. We analyzed 4342 proteins deduced from the Schistosoma mansoni transcriptome with bioinformatic computer programs. Thirty-four proteins had membrane-bound motifs. Within this group, we selected the Sm29 protein to be further characterized by in silico analysis. Sm29 was found to have a signal peptide made up of 26 amino acids, with a cleavage site between Ser26 and Val27. The glycosylation site search revealed three threonines (39, 132 and 133) with high probability of O-glycosylation and two asparagines (58 and 115) with high probability of N-glycosylation. Only one transmembrane helix was found in the C-terminal region of the protein from Leu169 to Lis191. The search for similarities and conserved motifs show that Sm29 is a protein with high identity to proteins present in S. japonicum (53, 52, 49, and 37% of identity) and it possesses disulfide-rich conserved domains. Apparently, Sm29 is a membrane bound protein, and it may be an important molecule in host-parasite interactions.

Avaliação do peptídeo H da proteína Sm14 na infecção experimental por Schistosoma mansoni

O peptídeo estudado neste trabalho (peptídeo H) corresponde a uma seqüência de aminoácidos entre a posição 15aa e 35aa da proteína Sm14 e foi avaliado na sua configuração original (pepH) e alterada com a inclusão de resíduos de cisteína nas extremidades amino e carboxila terminal (pepH-C1 e pepH-C2). Camundongos suiços foram imunizados com os peptídeos e posteriormente desafiados com cercárias de S. mansoni. Os ensaios imunoenzimáticos (ELISA) revelaram que o grupo de animais imunizados com pepH ou pepH-C2 associados ao adjuvante completo/incompleto de Freund, apresentaram níveis significativos de anticorpos específicos contra o peptídeo e contra a proteína Sm14r. A análise da especificidade através do Western Blotting mostrou que os anticorpos IgG presentes nos soros destes animais reconheceram o peptídeo e a proteína, sugerindo que a conformação adotada pelo peptídeo H mantêm certa homologia com a conformação na estrutura nativa da molécula. O mesmo resultado não foi observado para o grupo de animais imunizados com o pepH-C1 associado ao adjuvante. A avaliação das subclasses presentes nos soros dos animais imunizados com os peptídeos H e H-C2 associados ao adjuvante, mostrou expressão de níveis semelhantes de IgG1, IgG2a e IgG3, sugerindo que um perfil de resposta do tipo ThO foi estimulada. Os peptídeos H e H-C2 associados ao adjuvante se mostraram antigênicos

No entanto, a análise de proteção mostrou que não ocorreu redução da carga de vermes. A presença ou não dos resíduos de cisteína nas extremidades destes peptídeos parece não ter interferido na questão da proteção, apesar do estudo teórico apontar para diferenças ocorridas no que diz respeito a distribuição de cargas e na distância entre a luz das hélices que constituem o peptídeo. Contudo, para aqueles animais imunizados somente com o pepH-C1, uma redução significativa de 25 por cento foi observada. O peptídeo H também foi avaliado quanto à capacidade de interferir na formação do granuloma hepático. No entanto, o estudo histopatológico mostrou que imunizações utilizando o peptídeo não induziram alterações na reação granulomatosa em torno do ovo de S.mansoni.Sendo o S.mansoni um parasito de grande complexidade imunológica, a resposta protetora deve exigir um número maior de diferentes epítopos com diferentes especificidades de anticorpos.Assim,a construção de antígenos peptídeos múltiplos(MAP)com epítopos da proteína Sm14,seria mais um caminho a ser trilhado na busca pela vacina contra a esquistossomose mansoni

Avaliação do peptídeo H da proteína Sm14 na infecção experimental por Schistosoma mansoni / Evaluation of peptide H protein Sm14 in experimental infection with Schistosoma mansoni

O peptídeo estudado neste trabalho (peptídeo H) corresponde a uma seqüência de aminoácidos entre a posição 15aa e 35aa da proteína Sm14 e foi avaliado na sua configuração original (pepH) e alterada com a inclusão de resíduos de cisteína nas extremidades amino e carboxila terminal (pepH-C1 e pepH-C2). Camundongos suiços foram imunizados com os peptídeos e posteriormente desafiados com cercárias de S. mansoni. Os ensaios imunoenzimáticos (ELISA) revelaram que o grupo de animais imunizados com pepH ou pepH-C2 associados ao adjuvante completo/incompleto de Freund, apresentaram níveis significativos de anticorpos específicos contra o peptídeo e contra a proteína Sm14r. A análise da especificidade através do Western Blotting mostrou que os anticorpos IgG presentes nos soros destes animais reconheceram o peptídeo e a proteína, sugerindo que a conformação adotada pelo peptídeo H mantêm certa homologia com a conformação na estrutura nativa da molécula. O mesmo resultado não foi observado para o grupo de animais imunizados com o pepH-C1 associado ao adjuvante. A avaliação das subclasses presentes nos soros dos animais imunizados com os peptídeos H e H-C2 associados ao adjuvante, mostrou expressão de níveis semelhantes de IgG1, IgG2a e IgG3, sugerindo que um perfil de resposta do tipo ThO foi estimulada. Os peptídeos H e H-C2 associados ao adjuvante se mostraram antigênicos. No entanto, a análise de proteção mostrou que não ocorreu redução da carga de vermes. A presença ou não dos resíduos de cisteína nas extremidades destes peptídeos parece não ter interferido na questão da proteção, apesar do estudo teórico apontar para diferenças ocorridas no que diz respeito a distribuição de cargas e na distância entre a luz das hélices que constituem o peptídeo. Contudo, para aqueles animais imunizados somente com o pepH-C1, uma redução significativa de 25 por cento foi observada. O peptídeo H também foi avaliado quanto à capacidade de interferir na formação do granuloma hepático. No entanto, o estudo histopatológico mostrou que imunizações utilizando o peptídeo não induziram alterações na reação granulomatosa em torno do ovo de S.mansoni. Sendo o S.mansoni um parasito de grande complexidade imunológica, a resposta protetora deve exigir um número maior de diferentes epítopos com diferentes especificidades de anticorpos. Assim,a construção de antígenos peptídeos múltiplos (MAP) com epítopos da proteína Sm14, seria mais um caminho a ser trilhado na busca pela vacina contra a esquistossomose mansoni.

Preliminary analysis of Sm14 in distinct fractions of Schistosoma mansoni adult worm extract

In previous studies it was shown that the recombinant molecule, r-Sm14, induces high levels of protection against Schistosoma mansoni infection in two outbred animal models and immune crossprotection against infection by Fasciola hepatica in Swiss outbred mice. r-Sm14 was derived from a living worm extract, called SE, and is being developed as the molecular basis of an anti-helminth bivalent vaccine against the two parasites, for medical and veterinary application. Present data refer to SDS-PAGE and Western Blotting analysis of four different preparations of S. mansoni adult worms focusing Sm14 identification. The extracts correspond to the initial fraction of the SE extraction process, containing products released by living worms (SEi); SE2, reextraction of adult worms in PBS; and SE of separated male and female adult worms. In all extracts it was possible to detect the component of 14 kDa, that was recognized by specific anti-rSm14 antibody raised in rabbits

Ultrastructural and biochemical detection of biotin and biotinylated polypeptides in Schistosoma mansoni

Biotinylation is proposed for the identification of surface proteins in Schistosoma mansoni using the streptavidin-HRP conjugate for the detection of labeled polypeptides. However, control samples also showed several endogenous biotinylated polypeptides. In an attempt to determine the possibility of monspecific binding between the streptavidin-HRP conjugate and polypeptides from S. mansoni, the conjugate was blocked with biotinamidecaproate-N-hydroxysuccinimide ester (BcapNHS) before biotin-streptavidin blotting. No bands were detected on the nitrocellulose sheet, demonstrating the especific recognition of biotin by the streptavidin present in the conjugate. Whole cercariae and cercarial bodies and tails showed several endogenous biotinylated polypeptides. The biotin concentration was 13 mug/190,000 cercariae. Adult worms presented less endogenous biotinylated polypeptides than cercariae. These results may be due to changes in the environment from aerobic to anaerobic conditions when cercarial bodies (schistosomula) are transformed into adult worms and a decrease in CO2 production may occur. Cercariae, cercarial bodies and adult male worms were examined by transmission electron microscopy employing an avidin-colloidal gold conjugate for the detection of endogenous biotin. Gold particles were distributed mainly on the muscle fibers, but dispersed granules were observed in the tegument, mitochondria and cytosol. The discovery of endogenous biotin in S. mansoni should be investigate in order to clarify the function of this vitamin in the parasite.

Identification and characterization of sex-linked proteins of Schistosoma mansoni

The proteins of adults worms (male and female) of two isolates (BH and RJ) of Shistosoma mansoni were extracted using Triton X-114 phase separation. The SDS-polyacrilamide gel electrophoresis profiles of the three phases (detergent, aqueous and insoluble proteins) obtained were compared after Coomassie blue and silver staining, surface radioiodination and Western blotting. No major differences were detected between the 2 isolates. Of the 25 or more proteins which partitioned into the detergent phase, only about 8 proteins could be surface radiodinated on live adult worms. A comparison was also made between the profiles of mael and females worms, isolated from bisexually infected mice. Two major female-specific and one male-specific band were detected by silver and/or Coomassie staining. The female bands, 32 KDa and 18 Kda, partitioned into the detergent and aqueous phase, respectively. The male-specific band of 42 KDa remained in the insoluble phase. Antigenic differences between male and females protins were detected by Western vlotting using a sera from infected Nectomys squamipes