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

CA2+/H+ EXCHANGE IN ACIDIC VACUOLES OF TRYPANOSOMA-BRUCEI

The use of digitonin to permeabilize the plasma membrane of Trypanosoma brucei procyclic and bloodstream trypomastigotes allowed the identification of a non-mitochondrial nigericin-sensitive Ca2+ compartment. The proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was able to cause Ca2+ release from this compartment, which was also sensitive to sodium orthovanadate. Preincubation of the cells with the vacuolar H+-ATPase inhibitor bafilomycin A(1) greatly reduced the nigericin-sensitive Ca2+ compartment. Bafilomycin A(1) inhibited the initial rate of ATP-dependent non-mitochondrial Ca2+ uptake and stimulated the initial rate of nigericin-induced Ca2+ release by permeabilized procyclic trypomastigotes. ATP-dependent and bafilomycin A(1)- and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl)-sensitive Acridine Orange uptake was demonstrated in permeabilized cells. Under these conditions Acridine Orange was concentrated in abundant cytoplasmic round vacuoles by a process inhibited by bafilomycin A(1), NBD-Cl, nigericin, and Ca2+. Vanadate or EGTA significantly increased Acridine Orange uptake, while Ca2+ released Acridine Orange from these preparations, thus suggesting that the dye and Ca2+ were being accumulated in the same acidic vacuole. Acridine Orange uptake was reversed by nigericin, bafilomycin A(1) and NH4Cl. The results are consistent with the presence of a Ca2+/H+-ATPase system pumping Ca2+ into an acidic vacuole, that we tentatively named the acidocalcisome.304122723

ENERGIZATION-DEPENDENT CA-2+ ACCUMULATION IN TRYPANOSOMA-BRUCEI BLOOD-STREAM AND PROCYCLIC TRYPOMASTIGOTES MITOCHONDRIA

The permeabilization of Trypanosoma brucei procyclic and bloodstream trypomastigotes with digitonin permitted the quantitative estimation of a mitochondrial membrane potential of the order of 130-140 mV, in both forms, using safranine O. Dependence on substrate oxidation and response of the procyclic mitochondrial membrane potential to phosphate, FCCP, valinomycin, and Ca2+ indicate that these mitochondria behave similarly to vertebrate mitochondria regarding the properties of their electrochemical proton gradient. In contrast, in bloodstream mitochondria, development of a membrane potential was independent of substrate oxidation and dependent on hydrolysis of ATP by the mitochondrial oligomycin-sensitive ATPase, as demonstrated by collapse of the membrane potential by oligomycin and its insensitivity to the respiratory chain-inhibitor antimycin A. Mitochondria of T. brucei bloodstream forms were also able to take up Ca2+ by an electrophoretic mechanism. This is the first report of the presence of a Ca2+ transport mechanism in an eukaryotic cell devoid of complete tricarboxylic acid cycle and respiratory chain, the activities of which are known to be regulated by changes in intramitochondrial calcium concentration in other cells.56225125

CALCIUM HOMEOSTASIS IN PROCYCLIC AND BLOOD-STREAM FORMS OF TRYPANOSOMA-BRUCEI - LACK OF INOSITOL 1,4,5-TRISPHOSPHATE-SENSITIVE CA2+ RELEASE

When Trypanosoma brucei procyclic trypomastigotes were permeabilized with digitonin in a reaction medium containing MgATP, succinate, and 3.5-mu-M free Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level (0-05-0.1-mu-M), a range that correlates favorably with that detected in the intact cells with fura-2. The carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive Ca2+ uptake, certainly represented by the endoplasmic reticulum, was completely inhibited by 500-mu-M vanadate. When vanadate instead of carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present, the Ca2+ set point was increased to 0.6-0.7-mu-M. The succinate dependence and carbonyl cyanide p-trifluoromethoxyphenylhydrazone sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. When bloodstream trypomastigotes were used, neither succinate nor alpha-glycerophosphate stimulated the mitochondrial Ca2+ uptake. The mitochondrial Ca2+ transport could be measured only in the presence of ATP and 500-mu-M vanadate to inhibit the endoplasmic reticulum uptake. Bloodstream trypomastigotes have a lower cytosolic Ca2+ Concentration, as detected with fura-2 and a smaller extramitochondrial Ca2+ pool than procyclic trypomastigotes. Despite the presence of inositol phosphates, as determined by [H-3]inositol incorporation, and the large extramitochondrial Ca2+ pool of procyclic trypomastigotes (61.7 nmol of Ca2+/mg of protein), no inositol 1,4,5-trisphosphate-sensitive Ca2+ release could be detected in these parasites.26796020602

EFFECT OF THAPSIGARGIN ON CALCIUM HOMEOSTASIS IN TRYPANOSOMA-CRUZI TRYPOMASTIGOTES AND EPIMASTIGOTES

By using the fluorescent calcium indicator fura-2, it was found that the concentration of free Ca2+ in the cytoplasm of Trypanosoma cruzi trypomastigotes incubated in the presence or absence of external calcium was maintained at very low levels (10-20 nM). When trypomastigotes were incubated in the presence of succinate and ATP and permeabilized with digitonin, they lowered the medium calcium concentration to a submicromolar level. In the presence of 1 muM FCCP the initial rate of Ca2+ sequestration by these permeabilized cells was very slow. When succinate alone was present, the initial rate of Ca2+ accumulation was slower than with ATP plus succinate, and the calcium set point was about 0.6 muM. The succinate dependence and FCCP sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. High concentrations of the tumor promoter thapsigargin slightly increased cytosolic Ca2+ in the presence of extracellular Ca2+ but had no effect on the FCCP- and oligomycin/antimycin A-insensitive Ca2+ pool. In addition, when used at those concentrations (4-20 muM), thapsigargin was shown to release Ca2+ from the mitochondria and to decrease the inner mitochondrial membrane potential of trypomastigotes and epimastigotes as measured using safranine O. Despite the presence of inositol phosphates as determined by [H-3]inositol incorporation, no IP3-sensitive Ca2+ release could be detected in trypomastigotes.59230531

CALCIUM HOMEOSTASIS IN TRYPANOSOMA-CRUZI AMASTIGOTES - PRESENCE OF INOSITOL PHOSPHATES AND LACK OF AN INOSITOL 1,4,5-TRISPHOSPHATE-SENSITIVE CALCIUM POOL

The permeabilization of Trypanosoma cruzi amastigotes with digitonin allowed the study of Ca2+ fluxes between intracellular organelles in situ. In addition, fura-2 was used to determine the cytosolic Ca2+ concentration in the intact cells. When amastigotes were permeabilized in a reaction medium containing MgATP, succinate and 3.5-mu-M Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level, a range which correlates favorably with that detected in the intact cells with fura-2. The presence of 1-mu-M FCCP strongly decreased the initial rate of Ca2+ sequestration by these permeabilized cells. This FCCP-insensitive Ca2+ uptake, probably represented by the endoplasmic reticulum, was completely inhibited by 500-mu-M vanadate. On the other hand, when vanadate instead of FCCP was present, the initial rate of Ca2+ accumulation was decreased and the Ca2+ set point was increased to about 0.8-mu-M. The succinate dependence and FCCP sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. Despite the presence of inositol phosphates, as determined by [H-3]inositol incorporation, and of a large extramitochondrial Ca2+ pool, no IP3-sensitive or thapsigargin-sensitive Ca2+ release could be detected in either amastigotes or epimastigotes.52225126

Respiration and oxidative phosphorylation in the apicomplexan parasite Toxoplasma gondii

Respiration, oxidative phosphorylation, and the mitochondrial membrane potential (Delta Psi) of tachyzoites of the apicomplexan parasite Toxoplasma gondii were assayed in situ using very low concentrations of digitonin to render their plasma membrane permeable to succinate, ADP, safranin O, and other small molecules. The rate of basal respiration was slightly increased by digitonin when the cells were incubated in medium containing succinate. ADP promoted an oligomycin-sensitive transition from resting to phosphorylating respiration. Respiration was sensitive to antimycin A and cyanide, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was oxidized by antimycin A-poisoned mitochondria. The addition of ADP after TMPD/ascorbate also resulted in phosphorylating respiration. The antitoxoplasmosis drug atovaquone, at a very low concentration (0.03 mu M), totally inhibited respiration and disrupted the mitochondrial membrane potential. Atovaquone was shown to inhibit the respiratory chain of T. gondii and mammalian mitochondria between cytochrome b and c(1) as occurs with antimycin A(1). Phosphorylation of ADP could not be obtained in permeabilized tachyzoites in the presence of either pyruvate, 3-oxo-glutarate, glutamate, isocitrate, dihydroorotate, alpha-glycerophosphate, or endogenous substrates. Although ADP phosphorylation was detected in the presence of malate, this activity was rotenone-insensitive and was probably due to the conversion of malate into succinate through a fumarate reductase activity that was detected in mitochondrial extracts. Together these results provide the first direct biochemical evidence that the respiratory chain and oxidative phosphorylation are functional in apicomplexan parasites, although the terminal respiratory pathway is different from that in the mammalian host.27347310403104