Michelle de Lima Detoni

Antimicrobial activity of recombinant Pg-AMP1, a glycine-rich peptide from guava seeds

Antimicrobial peptides (AMPs) are compounds that act in a wide range of physiological defensive mechanisms developed to counteract bacteria, fungi, parasites and viruses. These molecules have become increasingly important as a consequence of remarkable microorganism resistance to common antibiotics. This report shows Escherichia coli expressing the recombinant antimicrobial peptide Pg-AMP1 previously isolated from Psidium guajava seeds. The deduced Pg-AMP1 open reading frame consists in a 168 bp long plus methionine also containing a His6 tag, encoding a predicted 62 amino acid residue peptide with related molecular mass calculated to be 6.98 kDa as a monomer and 13.96 kDa at the dimer form. The recombinant Pg-AMP1 peptide showed inhibitory activity against multiple Gram-negative (E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Staphylococcus epidermides) bacteria. Moreover, theoretical structure analyses were performed in order to understand the functional differences between natural and recombinant Pg-AMP1 forms. Data here reported suggest that Pg-AMP1 is a promising peptide to be used as a biotechnological tool for control of human infectious diseases.

Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydro-lase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenbergs envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.

Occurrence of a conserved domain in ATP diphosphohydrolases from pathogenic organisms associated to antigenicity in human parasitic diseases

A polypeptide (r78-117) belonging to the potato apyrase was identified as a conserved domain shared with apyrase-like proteins from distinct pathogenic organisms, and was obtained as a 6xHis tag polypeptide (r-Domain B). By ELISA, high IgG, and IgG1 and IgG2a subtypes levels were detected in BALB/c mice pre-inoculated with r-Domain B. In Schistosoma mansoni adult worm or Leishmania (V.) braziliensis promastigote preparation, anti-r-Domain B antibodies inhibit 22-72% of the phosphohydrolytic activities and when immobilized on Protein A-Sepharose immunoprecipitate 42-91% of them. Western blots of the immunoprecipitated resin-antibody-antigen complexes identified bands of mw similar to those predicted for parasite proteins. Total IgG and subclasses of patients with leishmaniasis or schistosomiasis exhibited cross-immunoreactivity with r-Domain B. Therefore, the domain B within both S. mansoni SmATPDase 2 (r156-195) and L. (V.) braziliensis NDPase (r83-122) are potentially involved in the host immune response, and also seem to be conserved during host and parasites co-evolution.

Protein content and electrophoretic profile of insect galls on susceptible and resistant host plants of Bauhinia brevipes Vogel (Fabaceae)

Gall induction, mediated by insect-herbivore chemical stimuli, is the result from anatomical and biochemical alterations in the host-plant tissues that provides shelter, food and defence against natural enemies and the harsh environment to the gall inducer. Schizomya macrocapillata Maia (Diptera, Cecidomyiidae) induces galls on Bauhinia brevipes Vogel (Fabaceae); the galls are spherical, with long reddish hairs that cover the adaxial wall surface of the gall, and a protuberance is observed on the abaxial leaf surface. Some plants are resistant to gall formation and, in many cases, this formation is inhibited by hypersensitive reaction. In the present work, samples from different parts of the non-galled and galled tissues from resistant and susceptible plants were carefully dissected. Indicating elevated metabolic activity, the protein concentration was 1.5–4.5-fold higher in the abaxial portion of the galls than in any other tissues, regardless of whether the galls were from resistant or susceptible plants. Different tissues from susceptible and resistant plants had distinct protein concentrations, and the fractionation of the proteins by SDS–PAGE and silver-staining showed shared and/or specific polypeptides. We hypothesise that specific proteins, possibly from distinct metabolic pathways, are involved in the physiological processes that determine whether the plant shows total and/or partial host resistance to the galling-insect attack.

Leishmania (Viannia) braziliensis nucleoside triphosphate diphosphohydrolase (NTPDase 1): localization and in vitro inhibition of promastigotes growth by polyclonal antibodies

Nucleoside triphosphate diphosphohydrolase (NTPDase) activity was recently characterized in Leishmania (Viannia) braziliensis promastigotes (Lb), and an antigenic conserved domain (r82-121) from the specific NTPDase 1 isoform was identified. In this work, mouse polyclonal antibodies produced against two synthetic peptides derived from this domain (LbB1LJ, r82-103; LbB2LJ, r102-121) were used. The anti-LbB1LJ or anti-LbB2LJ antibodies were immobilized on protein A-sepharose and immunoprecipitated the NTPDase 1 of 48 kDa and depleted approximately 40% of the phosphohydrolytic activity from detergent-homogenized Lb preparation. Ultrastructural immunocytochemical microscopy identified the NTPDase 1 on the parasite surface and in its subcellular cytoplasmic vesicles, mitochondria, kinetoplast and nucleus. The ATPase and ADPase activities of detergent-homogenized Lb preparation were partially inhibited by anti-LbB1LJ antibody (43-79%), which was more effective than that inhibition (18-47%) by anti-LbB2LJ antibody. In addition, the immune serum anti-LbB1LJ (67%) or anti-LbB2LJ (33%) was cytotoxic, significantly reducing the promastigotes growth in vitro. The results appoint the conserved domain from the L. braziliensis NTPDase as an important target for inhibitor design and the potential application of these biomolecules in experimental protocols of disease control.

An antigenic domain within a catalytically active Leishmania infantum nucleoside triphosphate diphosphohydrolase (NTPDase 1) is a target of inhibitory antibodies

We identified a shared B domain within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83-122). By immunoprecipitation assays and Western blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87-99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.

Differential biochemical responses of Calliandra brevipes (Fabaceae, Mimosoidae) to galling behaviour by Tanaostigmodes ringueleti and T. mecanga (Hymenoptera, Tanaostigmatidae)

Two species of Tanaostigmodes, T. ringueleti and T. mecanga, induce two distinct gall morphotypes in Calliandra brevipes Benth. (Fabaceae: Mimosoidae), namely a globose and a fusiform one. Secondary and primary metabolism of the two galls was compared with that of the stem tissue of the host plant. Phytochemical screening of gall samples revealed that triterpenoids were exclusive of the globose gall, and sterols exclusive of the fusiform gall, whereas saponins were absent in both galls. Flavonoid content in the globose gall was significantly lower than that in the fusiform gall. As expected, high antioxidant activity was observed in the fusiform gall, which was associated with the high flavonoid content. Protein analyses showed the presence of specific polypeptides in globose (97, 75, 34 kDa) and fusiform (40, 33 kDa) galls. Sucrose, glucose and fructose contents were 1.4–3.3 times higher in the globose-gall than in non-galled tissue, whereas in the fusiform gall, fructose content was 2-fold increased. The interactions between the host and the two Tanaostigmodes showed both similarities and differences between them, and with the non-galled tissue. Taken together, the results suggested that the two gall inducers co-inhabiting C. brevipes are capable of manipulating the primary and secondary metabolism differentially for their own benefit and, thus, the nutritive hypothesis was reinforced.

Seasonal variation of phenolic content in galled and non-galled tissues of Calliandra brevipes Benth (Fabaceae: Mimosoidae)

(Seasonal variation of phenolic content in galls and non-galled tissues of Calliandra brevipes Benth (Fabaceae: Mimosoidae)). Two species, Tanaostigmodes ringueleti and T. mecanga, induce distinct galls on Calliandra brevipes Benth (Fabaceae: Mimosoidae), a globose and a fusiform gall morphotype. Seasonal changes of phenolic content in the tissues of the two distinct galls were compared to those of non-galled leaves and stems of the host plants over one year. Th e variation in the phenolic content profi les was similar in both non-galled and galled tissues, and was primarily associated with changes in the levels of rainfall, indicating a direct response to hydric stress. In periods of drastic changes in water precipitation, the alterations were signifi cantly higher in non-galled than in galled tissues suggesting that the gall inducers might limit the variation in the phenolic concentration for their own benefi t.

Galls from Calliandra brevipes BENTH (Fabaceae : Mimosoidae): evidence of apyrase activity contribution in a plant–insect interaction

By western blots, cross-immunoreactivity with polyclonal anti-potato apyrase antibodies identified the Calliandra brevipes apyrase as a band of 75 kDa in the tissues of non-galled stem and leaves, and those of globose galls. The non-galled tissues hydrolysed either ATP, ADP, UDP, GTP or GDP. In globose galls, ADP, UDP and GDP hydrolysis were 1.7–5.1-fold higher than in non-galled tissues. ADP and UDP hydrolysis either from non-galled or globose gall tissues were 10–38% stimulated by 5 mM calcium, and drastically reduced (66–99%) by the addition of 5 mM EDTA or EGTA, confirming the divalent cation dependence. Nucleotidase, phosphatase or ATPase activities contributed in lower reaction rates. Apyrase activity was confirmed in C. brevipes tissues by nondenaturing polyacrylamide gel electrophoresis and western blots. By histochemical techniques, the ADPase activity was found as a granular-dense lead phosphate deposit homogeneously distributed at the external surface, and inside the nutritive cells of the globose gall. The sites of polyclonal anti-potato apyrase antibodies corroborate these localisations. The globose galls of the C. brevipes stems increase their capacity of hydrolysing nucleotides, which could be associated with carbohydrate biosynthesis, signalling and/or cell proliferation, crucial for feeding and survival of the insect.