Role of GP82 in the Selective Binding to Gastric Mucin during Oral Infection with Trypanosoma cruzi

Oral infection by Trypanosoma cruzi has been the primary cause of recent outbreaks of acute Chagas' diseases. This route of infection may involve selective binding of the metacyclic trypomastigote surface molecule gp82 to gastric mucin as a first step towards invasion of the gastric mucosal epithelium and subsequent systemic infection. Here we addressed that question by performing in vitro and in vivo experiments. A recombinant protein containing the complete gp82 sequence (J18), a construct lacking the gp82 central domain (J18*), and 20-mer synthetic peptides based on the gp82 central domain, were used for gastric mucin binding and HeLa cell invasion assays, or for in vivo experiments. Metacyclic trypomastigotes and J18 bound to gastric mucin whereas J18* failed to bind. Parasite or J18 binding to submaxillary mucin was negligible. HeLa cell invasion by metacyclic forms was not affected by gastric mucin but was inhibited in the presence of submaxillary mucin. Of peptides tested for inhibition of J18 binding to gastric mucin, the inhibitory peptide p7 markedly reduced parasite invasion of HeLa cells in the presence of gastric mucin. Peptide p7*, with the same composition as p7 but with a scrambled sequence, had no effect. Mice fed with peptide p7 before oral infection with metacyclic forms developed lower parasitemias than mice fed with peptide p7*. Our results indicate that selective binding of gp82 to gastric mucin may direct T. cruzi metacyclic trypomastigotes to stomach mucosal epithelium in oral infection

Effects of estrogen status in osteocyte autophagy and its relation to osteocyte viability in alveolar process of ovariectomized rats

Estrogen maintains osteocyte viability, whereas its deficiency induces osteocyte apoptosis. As autophagy is important for osteocyte viability, we hypothesized whether the anti-apoptotic effect of estrogen is related to autophagy in osteocytes. Thirty adult female rats were sham-operated (SHAM) or ovariectomized (OVX). After three weeks, twelve rats of SHAM and OVX groups were killed before treatment (basal period), whereas the remaining rats received estrogen (OVXE) or vehicle (OVX) for 45 days. Fragments of maxilla containing alveolar process of the first molars were embedded in paraffin or Araldite. Paraffin-sections were stained with hematoxylin/eosin for histomorphometry, or subjected to the silver impregnation method for morphological analysis of osteocyte cytoplasmic processes. Autophagy was analyzed by immunohistochemical detections of beclin-1, MAP-LC3 alpha and p62, whereas apoptosis was evaluated by immunohistochemical detections of cleaved caspase-3 and BAX, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) method and by ultrastructural analysis. Araldite-semithin sections were subjected to the Sudan-black method for detection of lipids. OVX-basal group showed high frequency of caspase-3-, TUNEL- and p62-positive osteocytes accompanied with low frequency of beclin-1- and MAP-LC3 alpha-positive osteocytes. At 45 days, OVXE group exhibited higher number of osteocytes, higher frequency of beclin-1- and MAP-LC3 alpha-positive osteocytes, and lower frequency of caspase-3, BAX-, TUNEL- and p62-positive osteocytes than OVX group. Significant reduction in bone area was observed in the OVX compared to OVXE and SHAM groups. The highest frequency of Sudan-Black-positive osteocytes and osteocytes with scarce cytoplasmic processes, or showing apoptotic features were mainly observed in OVX groups. Our results indicate that estrogen deficiency decreases autophagy and increases apoptosis, whereas estrogen replacement enhances osteocyte viability by inhibiting apoptosis and maintaining autophagy in alveolar process osteocytes. These results suggest that the anti-apoptotic effect of estrogen may be, at least in part, related to autophagy regulation in osteocytes.Sao Paulo Research Foundation (FAPESP)National Council for Scientific and Technological Development (CNPq), BrazilCoordination of Improvement of Higher Level Personnel (CAPES), BrazilUniv Fed Sao Paulo UNIFESP, EPM, Dept Morfol & Genet, Disciplina Histol & Biol Estrutural, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, EPM, Dept Ginecol, Sao Paulo, SP, BrazilSao Paulo State Univ UNESP, Sch Dent, Araraquara Lab Histol & Embryol, Araraquara, SP, BrazilUniv Fed Sao Paulo UNIFESP, EPM, Dept Morfol & Genet, Disciplina Histol & Biol Estrutural, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, EPM, Dept Ginecol, Sao Paulo, SP, BrazilFAPESP: 2012/19428-8, 2012/22666-8Web of Scienc

Selective binding of <i>T. cruzi</i> metacyclic trypomastigotes to gastric mucin.

<p>A) Microtiter plates coated with varying amounts of gastric or submaxillary mucin were incubated with metacyclic forms for 1 h and processed for detection of bound parasites by ELISA. Values are the means of triplicates of one representative assay out of three. Variation between triplicates was <5%. B) Microtiter plates coated with gastric (G) or submaxillary (S) mucin (10 µg/well) were processed for ELISA using antibodies specific for gastric or submaxillary mucin at 1∶100 dilution. Values are the means of triplicates (variation <5%). C) Metacyclic forms were added to microtiter plates coated with gastric mucin (10 µg/well) and incubated in absence or in the presence of the indicated amounts of the recombinant protein J18 or GST and processed for ELISA. Values are the means of triplicates. (variation <10%). D) Metacyclic trypomastigotes were added to gastric mucin-coated coverslips and, following the procedure described in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000613#s2" target="_blank">methods</a> section, the Giemsa-stained parasites were visualized under the microscope. E) Transwell filters coated with gastric or submaxillary mucin were placed onto wells containing metacyclic forms. At different time points, samples from the filter chamber were collected and the number of parasites counted. Values represent the means ± standard deviation of three independent experiments. The difference in parasite translocation through gastric and submaxillary mucin layer was significant (*), with P<0.05. F) Gastric or submaxillary mucin was added to HeLa cells before addition of metacyclic trypomastigotes. After 1 h at 37°C, the cells were fixed and Giemsa-stained. The number of internalized parasites was counted in a total of 250 cells. Values correspond to means ± SD of 4 independent experiments. There was a significant difference between invasion in absence and in the presence of submaxillary mucin (*), P<0.01.</p

Inhibitory effect of peptide p7 on oral <i>T. cruzi</i> infection.

<p>A) Balb/c mice were divided in control (n = 5) and experimental (n = 5) groups, peptide p7* were given orally to control mice and peptide p7 to experimental animals 15 min before infection with metacyclic trypomastigotes by the oral route and the number of circulating parasites counted. Variations in the parasitemia levels between mice are indicated. B) Histological sections of the mouse stomach, collected 4 days after infection, were stained by hematoxylin and eosin and the number of amastigote nests (white arrow) was counted in 7 equivalent tissue sections. The representative results are shown, with bars corresponding to the variation in the number of parasites nests between sections.</p

Determination of <i>T. cruzi</i> gp82 sequences implicated in binding to gastric mucin.

<p>A) Amino acid composition of peptides corresponding to gp82 central domain. Shown are the 20-mer peptides with an overlap of 10 residues. B) Effect of peptides shown in (A) on J18 binding to gastric mucin. Values are the means of triplicates of representative assays (variation between triplicates <10%).</p

Specific binding of the recombinant protein J18 to gastric mucin through its central domain.

<p>A) The recombinant protein J18, containing the full length <i>T. cruzi</i> gp82 peptide sequence, was added to microtiter plates coated with gastric or submaxillary mucin at the indicated concentrations. Values are the means of triplicates of one representative assay out of three. Variation between triplicates was <5%. (B) C03, a recombinant protein of <i>T. cruzi</i> gp82 family with 59.1% identity with J18, was used for gastric mucin-binding assay. C) Gastric mucin preparations at pH 2.5 and pH 7.2 were used to coat microtiter plates and then binding of J18 was performed. In (B) and (C), the values are the means of triplicates (variation <5%). D) Schematic representation of recombinant proteins based on gp82 molecule. Shown are the GST-fused constructs containing the full-length gp82 sequence (J18) or lacking either the amino-terminal portion (J18b) or the central domain spanning residues 257–321 (J18*). E) Binding of J18, J18b and J18* to gastric mucin was compared. Values are the means of triplicates (variation <10%).</p

Peptide p7 inhibits host cell invasion by metacyclic trypomastigotes in the presence of gastric mucin.

<p>A) Parasites were incubated with HeLa cells in absence or in the presence of gastric mucin, plus the indicated peptides at 200 µg/ml. After 1 h incubation, the cells were fixed and stained with Giemsa for quantification of internalized parasites. Values are the means ± SD of 4 independent experiments. The inhibitory effect of peptide was significant (*) for p7, P = 0.005, and for p10, P<0.05. B). Metacyclic trypomastigotes were incubated with HeLa cells in the presence of gastric mucin, plus peptide p7 at the indicated concentrations and the reaction proceeded as above. Values are the means ± SD of 3 independent experiments. The difference in invasion rate in absence and in the presence of p7 was significant (*) at all concentrations, P<0.05. C) HeLa cells were incubated with metacyclic forms in the presence of gastric mucin, plus peptide p7 or p7* which has the same composition of p7 but a scrambled sequence. Values are the means ± SD of 4 independent assays performed in duplicate or triplicates.</p