Daily variations in the glycerol-3-phosphate dehydrogenase isoforms expression in Triatoma infestans flight muscles.

Triatoma infestans, the main vector of Chagas disease, is a blood-sucking insect. Flight dispersal of adults is the most important mechanism for reinfestation of houses after insecticide spraying. Flight muscles have two glycerol-3-phosphate dehydrogenase (GPDH) isoforms: GPDH-1 is involved in flight metabolism and GPDH-2 provides lipid precursors. In this study, we explored the profile of GPDH expression in females and males adult flight muscles under light/dark cycle, constant light, and constant dark conditions. Under constant dark conditions, GPDH-1 flight muscles of T. infestans showed a rhythmic pattern of transcription synchronous with a rhythmic profile of activity suggesting regulation by the endogenous circadian clock. Otherwise, the GPDH-2 expression analysis showed no regulation by the endogenous clock, but showed that an external factor, such as the dark/light period, was necessary for synchronization of GPDH-2 transcription and activity.

Differential expression of glycerol-3-phosphate dehydrogenase isoforms in flight muscles of the Chagas disease vector Triatoma infestans (Hemiptera, Reduviidae).

Flight muscles of Triatoma infestans have two glycerol-3-phosphate dehydrogenase (GPDH) isoforms: GPDH-1 is involved in flight metabolism and GPDH-2 provides lipid precursors. Total GPDH activity was greater in the natural population and almost only due to GPDH-1. Different expression and activity observed between GPDH isoforms in the natural population and the first laboratory generation was not detected in the second laboratory generation. This pattern may be caused by gradual adaptation to laboratory nutritional conditions. During development, the expression of GPDH-2 increased with a longer time of intake, which would imply an increment in lipid biosynthesis. The GPDH-1 transcript predominated with respect to that of GPDH-2 in the lower nutritional condition, suggesting the necessity of insects to fly during this nutritional status. The transcriptional pattern showed a delay at 22°C. The isoforms activities and transcript patterns in flight muscles suggest transcriptional adaptation to metabolic requirements originated by alternative splicing.

Gene discovery in Triatoma infestans.

BACKGROUND: Triatoma infestans is the most relevant vector of Chagas disease in the southern cone of South America. Since its genome has not yet been studied, sequencing of Expressed Sequence Tags (ESTs) is one of the most powerful tools for efficiently identifying large numbers of expressed genes in this insect vector. RESULTS: In this work, we generated 826 ESTs, resulting in an increase of 47% in the number of ESTs available for T. infestans. These ESTs were assembled in 471 unique sequences, 151 of which represent 136 new genes for the Reduviidae family. CONCLUSIONS: Among the putative new genes for the Reduviidae family, we identified and described an interesting subset of genes involved in development and reproduction, which constitute potential targets for insecticide development.

Differential tissue and flight developmental expression of glycerol-3-phosphate dehydrogenase isozymes in the Chagas disease vector Triatoma infestans.

Glycerol-3-phosphate dehydrogenase (GPDH) isozymes are differentially expressed among tissues and during flight development. GPDH-1 is involved in the flight-muscle metabolism and GPDH-2 provides precursors for lipid biosynthesis in many tissues. We have isolated and characterized from Triatoma infestans, a Chagas disease vector, two cDNAs encoding for GPDH-1 and GPDH-2 isozymes. The inferred amino acid sequences showed high identity with other GPDH sequences from flying insects. A GPDH-2 transcript was found in fifth instar nymphs, thoracic muscles, adult gonads, and fat bodies. Both isozymes are present in 30-day-old adult thoracic muscle transcripts, and the pattern of expression differs between sexes. The expression of GPDH-1 begins earlier in females, and GPDH-2 is expressed more abundantly in female adult thoracic muscles than in those from males. This finding is consistent with those of other investigators who showed a higher flight initiation probability in T. infestans females than in males.

Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human alpha(2)-macroglobulin and pregnancy zone protein.

Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as alpha2-macroglobulin (alpha2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of alpha2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both alpha-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native alpha2-M and PZP, respectively; (ii) limited proteolysis of bait region in alpha2-M and PZP, and (iii) covalent binding of cruzipain to PZP and alpha2-M. Conformational and structural changes experimented by alpha-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain-alpha-M complexes were also detected by gelatin SDS-PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, alpha2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, alpha-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human alpha-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on host's molecules and hypothetically regulate parasite functions controlled by cruzipain.