Phosphorus-31 NMR Spectroscopy of Parasitic Protozoa
31P NMR spectra have been obtained of various parasitic protozoa including Trypanosoma cruzi, Leishmania major, Trypanosoma brucei, Plasmodium berghei, Toxoplasma gondii, and Cryptosporidium parvum. The unifying features of solution-state 31P NMR spectra of acid extracts of these parasites were high levels of pyrophosphate, and in most cases similarly high levels of the condensed inorganic phosphates: tri-, tetra- and pentapolyphosphate. Spectra were obtained at high resolution and complete resonance assignments have been made. Further investigation by 31P NMR of the acidocalcisomes of these parasites indicated that inorganic phosphate and inorganic condensed phosphates were the sole contributors to the NMR spectra of the acid extracts of these organelles and permitted the calculation of the average chain length of the polyphosphates which was 3.11--3.39 phosphates per chain. In order to study intact, unextracted acidocalcisomal polyphosphates, organelles were isolated and subjected to solid-state 31P NMR magic-angle spinning experiments. Isotropic shifts were detected in spectra of acidocalcisomes from three species of trypanosome, corresponding to orthophosphate and alpha- (terminal) and beta- (bridging) phosphates of polyphosphate. Spinning sidebands of the latter two resonances permitted the calculation of the chemical shift tensor elements associated with the experimental spectra, which compared well with model compounds. Analysis of the relative intensities of the alpha- and beta-resonances in these spectra again allowed for the calculation of the average polyphosphate chain length, which was 3.05--3.59. In vivo solution-state 31P NMR spectra of suspensions of living trypanosomes failed to show large resonances corresponding to the alpha- and beta-phosphates of polyphosphate which was attributed to their motionally restricted, solid-like state within the acidocalcisomes. In the case of epimastigote forms of T. cruzi, in vivo 31P NMR spectra detected ∼mM levels of UDP-hexose compounds and further spectroscopic evidence supported the breakdown and synthesis of these compounds via enzymes which are not well characterized in trypanosomes. These results have identified metabolic pathways in these parasites which are utilized for novel functions by comparison to mammals, and may therefore represent targets for future parasite control.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign