Cathepsins L and B in Dysdercus peruvianus, Rhodnius prolixus, and Mahanarva fimbriolata. Looking for enzyme adaptations to digestion.

Cysteine peptidases (CP) play a role as digestive enzymes in hemipterans similar to serine peptidases in most other insects. There are two major CPs: cathepsin L (CAL), which is an endopeptidase and cathepsin B (CAB) that is both an exopeptidase and a minor endopeptidase. There are thirteen putative CALs in Dysdercus peruvianus, which in some cases were confirmed by cloning their encoding genes. RNA-seq data showed that DpCAL5 is mainly expressed in the anterior midgut (AM), DpCAL10 in carcass (whole body less midgut), suggesting it is a lysosomal enzyme, and the other DpCALs are expressed in middle (MM) and posterior (PM) midgut. The expression data were confirmed by qPCR and enzyme secretion to midgut lumen by a proteomic approach. Two CAL activities were isolated by chromatography from midgut samples with similar kinetic properties toward small substrates. Docking analysis of a long peptide with several DpCALs modeled with digestive Tenebrio molitor CAL (TmCAL3) as template showed that on adapting to luminal digestion DpCALs (chiefly DpCAL5) changed in relation to their ancestral lysosomal enzyme (DpCAL10) mainly at its S2 subsite. A similar conclusion arrived from structure alignment-based clustering of DpCALs based on structural similarity of the modeled structures. Changes mostly on S2 subsite could mean the enzymes turn out less peptide-bond selective, as described in TmCALs. R. prolixus CALs changed on adapting to luminal digestion, although less than DpCALs. Both D. peruvianus and R. prolixus have two digestive CABs which are expressed in the same extension as CALs, in the first digestive section of the midgut, but less than in the other midgut sections. Mahanarva fimbriolata does not seem to have digestive CALs and their digestive CABs are mainly expressed in the first digestive section of the midgut and do not diverge much from their lysosomal counterparts. The data suggest that CABs are necessary at the initial stage of digestion in CP-dependent Hemipterans, which action is completed by CALs with low peptide-bond selectivity in Heteroptera species. In M. fimbriolata protein digestion is supposed to be associated with the inactivation of sap noxious proteins, making CAB sufficient as digestive CP. Hemipteran genomes and transcriptome data showed that CALs have been recruited as digestive enzymes only in heteropterans, whereas digestive CABs occur in all hemipterans.

Knockdown of the Rhipicephalus microplus cytochrome c oxidase subunit III gene is associated with a failure of Anaplasma marginale transmission.

Rhipicephalus microplus is an obligate hematophagous ectoparasite of cattle and an important biological vector of Anaplasma marginale in tropical and subtropical regions. The primary determinants for A. marginale transmission are infection of the tick gut, followed by infection of salivary glands. Transmission of A. marginale to cattle occurs via infected saliva delivered during tick feeding. Interference in colonization of either the tick gut or salivary glands can affect transmission of A. marginale to naïve animals. In this study, we used the tick embryonic cell line BME26 to identify genes that are modulated in response to A. marginale infection. Suppression-subtractive hybridization libraries (SSH) were constructed, and five up-regulated genes {glutathione S-transferase (GST), cytochrome c oxidase sub III (COXIII), dynein (DYN), synaptobrevin (SYN) and phosphatidylinositol-3,4,5-triphosphate 3-phosphatase (PHOS)} were selected as targets for functional in vivo genomic analysis. RNA interference (RNAi) was used to determine the effect of tick gene knockdown on A. marginale acquisition and transmission. Although RNAi consistently knocked down all individually examined tick genes in infected tick guts and salivary glands, only the group of ticks injected with dsCOXIII failed to transmit A. marginale to naïve calves. To our knowledge, this is the first report demonstrating that RNAi of a tick gene is associated with a failure of A. marginale transmission.

Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum.

BACKGROUND: Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. METHODS: The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis-Menten; also, inhibition assays were performed using risedronate. RESULTS: The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 µM, 7.8 ± 1.3 µM and 2.06 ± 0.4 µM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. CONCLUSIONS: The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus.

The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106kDa (gel filtration) and 55kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, AßNA and AsnMCA (K(m)s 0.65, 0.14, 0.68 and 0.74mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3, Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species.

Host-mediated induction of alpha-amylases by larvae of the Mexican bean weevil Zabrotes subfasciatus (Coleoptera: Chrysomelidae: Bruchinae) is irreversible and observed from the initiation of the feeding period.

Larvae of Zabrotes subfasciatus secrete alpha-amylases that are insensitive to the alpha-amylase inhibitor found in seeds of Phaseolus vulgaris. By analyzing amylase activities during larval development on P. vulgaris, we detected activity of the constitutive amylase and the two inducible amylase isoforms at all stages. When larvae were transferred from the non alpha-amylase inhibitor containing seeds of Vigna unguiculata to P. vulgaris, the inducible alpha-amylases were expressed at the same level as in control larvae fed on P. vulgaris. Interestingly, when larvae were transferred from seeds of P. vulgaris to those of V. unguiculata, inducible alpha-amylases continued to be expressed at a level similar to that found in control larvae fed P. vulgaris continuously. When 10-day-old larvae were removed from seeds of V. unguiculata and transferred into capsules containing flour of P. vulgaris cotyledons, and thus maintained until completing 17 days (age when the larvae stopped feeding), we could detect higher activity of the inducible alpha-amylases. However, when larvae of the same age were transferred from P. vulgaris into capsules containing flour of V. unguiculata, the inducible alpha-amylases remained up-regulated. These results suggest that the larvae of Z. subfasciatus have the ability to induce insensitive amylases early in their development. A short period of feeding on P. vulgaris cotyledon flour was sufficient to irreversibly induce the inducible alpha-amylase isoforms. Incubations of brush border membrane vesicles with the alpha-amylase inhibitor 1 from P. vulgaris suggest that the inhibitor is recognized by putative receptors found in the midgut microvillar membranes.

Transporters involved in glucose and water absorption in the Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) anterior midgut.

Little is known about insect intestinal sugar absorption, in spite of the recent findings, and even less has been published regarding water absorption. The aim of this study was to shed light on putative transporters of water and glucose in the insect midgut. Glucose and water absorptions by the anterior ventriculus of Dysdercus peruvianus midgut were determined by feeding the insects with a glucose and a non-absorbable dye solution, followed by periodical dissection of insects and analysis of ventricular contents. Glucose absorption decreases glucose/dye ratios and water absorption increases dye concentrations. Water and glucose transports are activated (water 50%, glucose 33%) by 50 mM K(2)SO(4) and are inhibited (water 46%, glucose 82%) by 0.2 mM phloretin, the inhibitor of the facilitative hexose transporter (GLUT) or are inhibited (water 45%, glucose 35%) by 0.1 mM phlorizin, the inhibitor of the Na(+)-glucose cotransporter (SGLT). The results also showed that the putative SGLT transports about two times more water relative to glucose than the putative GLUT. These results mean that D. peruvianus uses a GLUT-like transporter and an SGLT-like transporter (with K(+) instead of Na(+)) to absorb dietary glucose and water. A cDNA library from D. peruvianus midgut was screened and we found one sequence homologous to GLUT1, named DpGLUT, and another to a sodium/solute symporter, named DpSGLT. Semi-quantitative RT-PCR studies revealed that DpGLUT and DpSGLTs mRNA were expressed in the anterior midgut, where glucose and water are absorbed, but not in fat body, salivary gland and Malpighian tubules. This is the first report showing the involvement of putative GLUT and SGLT in both water and glucose midgut absorption in insects.