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Plasma purines and Trypanosoma brucei

Samuel J. Black, Qin Wang, Erika Hamilton, Jun Wang, Andrew Van Praagh and Madhavi Muranjan

Dept. of Vet. and Animal Sciences. Univ. Massachusetts, Paige Lab. Amhherst MA 01003, U.S.A.


Acknowledgement
References

Trypanosoma brucei is an obligate auxotroph for purines (Hassan and Coombs, 1988). A bloodstream stage trypomastigote can salvage preformed oxypurines, purine nucleobases and purine nucleosides (James and Born, 1980; Carter and Fairlamb, 1993) and interconvert any of these into all of the cellular nucleotides (Fish et al., 1982). Purine interconversion allows replication of bloodstream stage T. brucei S 427 clone 1 in medium containing either, xanthine, hypoxanthine, adenine, guanine, adenosine or guanosine as the sole purine (Wang and Black, unpublished). When their purine supply is made growth-limiting T. brucei cease to replicate and accumulate in the first gap phase (G1) of the cell devision cycle (Hamilton and Black, unpublished). Our studies on the mechanism of innate resistance to trypanosomiasis in cape buffalo indicate that systemic purine catabolism may contribute to restrained trypanosome population growth in these reservoir hosts.

Cape buffalo are migrant wild bovids that co-evolved with trypanosomes and can inhabit regions of Africa where trypanosome transmission is high. Whereas trypanosomiasis is fatal in people and domestic animals, infected cape buffalo show no signs of disease. The cape buffalo contain a constitutive serum protein that kills all species of African trypanosomes in vitro (Reduth et al., 1994) or prevents their replication depending on assay buffer constituents. We have isolated the trypanocidal/trypanostatic component of cape buffalo serum and identified it as xanthine oxidase (Muranjan et al., submitted). Xanthine oxidase catabolizes hypoxanthine and xanthine to uric acid yielding hydrogen peroxide and superoxide anion as reaction byproducts. Trypanocidal activity is due to inhibition of trypanosome glycolysis by hydrogen peroxide generated during catabolism of extracellular oxypurine. Trypanostatic activity is revealed when trypanocidal hydrogen peroxide is inactivated by including pyruvate and/or catalase in the assay buffer, and results from growth-limiting purine.

To obtain information on substrate availability for systemic xanthine oxidase we used reverse phase HPLC on a C-18 column to assay purines and purine catabolic enzymes, in serum and plasma from cape buffalo and other mammals. There was a trace of inosine and/or guanosine (the nucleosides did not resolve) in cape buffalo serum (<3 micromolar concentration) and no other purine was detected (level of detection 1 micromolar). Freshly isolated cow plasma also contained a trace of inosine and/or guanosine, and freshly isolated mouse plasma contained a trace (< 3 micromolar concentration) of adenosine. Other purines were not detected in the fresh plasma samples. The purine content of mouse plasma was obtained using blood that was collected into an ice cold hematocrit tube for plasma isolation.

Recovered plasma was centrifuged at 4°C over an Amicon 3 membrane to remove purine catabolic enzymes and other molecules of >3 kDa, and the purine content of the filtrate was immediately characterized by reverse phase HPLC. The values ascribed for free plasma purines may be under-estimates as some purine catabolism may have occurred prior to HPLC analysis, however, in the case of mouse plasma, the error is likely to be small.

The cape buffalo serum contained purine nucleoside phosphorylase, adenosine deaminase, guanosine deaminase, and guanine deaminase in addition to xanthine oxidase (Q. Wang et al., unpublished). Freshly isolated cow and mouse plasma also contained purine catabolic enzymes although some of these were at lower concentrations than detected in the cape buffalo serum (E. Hamilton et al., unpublished). There was no indication of cell lysis during plasma preparation and the presence of purine catabolic enzymes in the preparations of plasma are unlikely to result from cell damage during blood collection.

The studies show that plasma purines are present at low concentration and are accompanied by several purine catabolic enzymes. Numerous questions remain: How do T. brucei trypomastigotes access enough purine to support their replication? Do trypanosomes induce purine flux into the blood plasma? Are there sequestered purines in plasma that are inaccessible to purine catabolic enzymes but accessible to trypanosomes? Do trypanosomes intercept and utilize the methylated and other modified purines that are typically filtered and excreted in urine and if so do they first remove the modifying groups? The parasites do have a transport site for highly modified purine which includes the trypanocidal drug MDL 73811 (Byers et al., 1992). What percentage of salvaged purine is released as oxypurine by trypanosomes? Is enough hydrogen peroxide generated on the trypanosome cell surface during catabolism of released oxypurine, to affect their replication in vivo? Does innate resistance to trypanosomiasis in cape buffalo result from systemic xanthine oxidase activity? Do susceptible mammals lack systemic xanthine oxidase, or do they have additional plasma components that inactivate reactive oxygen intermediates generated during catabolism of oxypurine by xanthine oxidase?

Acknowledgement

We thank the Kenya Agricultural Research Institute and the International Livestock Research Institute, Nairobi, Kenya for provision of cape buffalo serum. This research was supported by NIH 1 R01 Al 35646-01A2 TMP.

References

Byers, T.L., Casara, P. and Bitonti, A.J., 1992. Uptake of the antitrypanosomal drug 5'-([(Z)-4-amino-2-butenyl] methyamino)-5'-deoxyadenosine (MDL 73811) by the purine transport system of Trypanosoma brucei brucei. Biochem. J., 283: 755-758.

Carter, N.S. and Fairlamb, A.H., 1993. Arsenical-resistant trypanosomes lack an unusual adenosine transporter Nature (Lond.) 361: 173-176. Published erratum; Nature 1993, 361: 374.

Fish, W.R., Looker, D.L., Marr, J.J. and Berens, R.L., 1982. Purine metabolism in the bloodstream forms of Trypanosoma gambiense and T. rhodesiense. Biochim. Biophys. Acta., 719: 223-231.

Hassan, H.F. and Coombs, G.H., 1988. Purine and pyrimidine metabolism in parasitic protozoa. FEMS Microbiol. Rev., 45: 47-84.

James, D.M. and Born, G.V.R., 1980. Uptake of purine bases and nucleosides in African trypanosomes. Parasitol. 81: 383-393.

Reduth, D., Grootenhuis, J.G., Olubayo, R.O., Muranjan, M., Otieno-Omondi, F.P., Morgan, G.A., Brun, R., Williams, D.J.L. and Black, S.J., 1994. African buffalo serum contains novel trypanocidal protein. J. Euk. Microbiol., 41: 95-103.


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