Hydrolysis products of cAMP analogs cause transformation of Trypanosoma brucei from slender to stumpy-like forms

Abstract

African sleeping sickness is a disease caused by Trypanosoma brucei. T. brucei proliferate rapidly in the mammalian bloodstream as long, slender forms, but at higher population densities they transform into nondividing, short, stumpy forms. This is thought to be a mechanism adopted by T. brucei to establish a stable host-parasite relationship and to allow a transition into the insect stage of its life cycle. Earlier studies have suggested a role for cAMP in mediating this transformation. In this study, using membrane-permeable nucleotide analogs, we show that it is not the cAMP analogs themselves but rather the hydrolyzed products of membrane-permeable cAMP analogs that prevent proliferation of T. brucei. The metabolic products are more potent than the cAMP analogs, and hydrolysis-resistant cAMP analogs are not antiproliferative. We further show that the antiproliferative effect of these membrane-permeable adenosine analogs is caused by transformation into forms resembling short, stumpy bloodstream forms. These data suggest that the slender-to-stumpy transformation of T. brucei may not be mediated directly by cAMP and also raise the possibility of using such adenosine analogs as antitrypanosomal drugs.

Fig. 1.

8-pCPT-2′-O-Me-cAMP inhibits T. brucei PDE activity and causes a small increase in cAMP. (A) Inhibition of recombinant TbrPDEB1 holoenzyme (open triangles) or TbrPDEB2 holoenzyme (filled triangles) by 8-pCPT-2′-O-Me-cAMP. (B) Inhibition of bloodstream-form T. brucei lysate PDE activity by 8-pCPT-2′-O-Me-cAMP (1 μM). (C) Fold change in cAMP levels in bloodstream-form T. brucei after incubation for 90 min with 25 μM 8-pCPT-2′-O-Me-cAMP or 50 μM dipyridamole. (D) Inhibition of proliferation of bloodstream-form T. brucei by 8-pCPT-2′-O-Me-cAMP (circles), in the presence of 1 mM inosine (triangles, broken line) or 1 mM adenine (squares)

Fig. 2.

cAMP analogs show potent antiproliferative effects in bloodstream-form T. brucei, but hydrolysis-resistant cAMP analogs do not. (A) Inhibition of proliferation of bloodstream-form T. brucei (over 48 h) by 8-pCPT-2′-O-Me-cAMP or Sp-8-pCPT-2′-O-Me-cAMPS. (B) Inhibition of proliferation of bloodstream-form T. brucei by 8-pCPT-cAMP (squares), Sp-8-pCPT-cAMPS (triangles), or Sp-5,6-DCl-cBIMPS (circles).

Fig. 3.

Etazolate shows weak inhibition of T. brucei PDE activity but potent antiproliferative activity. Inhibition of PDE activity in bloodstream-form T. brucei lysates [1 μM (29)] (triangles, left y axis) and inhibition of proliferation of bloodstream-form T. brucei by etazolate (squares, right y axis).

Fig. 4.

8-pCPT-2′-O-Me-cAMP is hydrolyzed by trypanosomes in media, and hydrolysis products show potent, bloodstream-form-specific antiproliferative effects. (A) Relative amount of 8-pCPT-2′-O-Me-cAMP present in medium plus T. brucei over time, measured by using LC/MS (y axis cAMP = 8-pCPT-2′-O-Me-cAMP). (B) Relative amount of 8-pCPT-2′-O-Me-cAMP present in lysed T. brucei over time, measured by using LC/MS (y axis cAMP = 8-pCPT-2′-O-Me-cAMP). (C) Inhibition of proliferation of bloodstream-form T. brucei by 8-pCPT-2′-O-Me-5′-AMP (squares) or 8-pCPT-2′-O-Me-ado (triangles). (D) Inhibition of proliferation of bloodstream-form (strain 427) T. brucei (squares), T. evansi (triangles, broken line), or procyclic IsTar 1.7 T. brucei by 8-pCPT-2′-O-Me-5′-AMP (circles) or by 8-pCPT-2′-O-Me-cAMP (diamonds).

Fig. 5.

8-pCPT-2′-O-Me-5′-AMP converts monomorphic bloodstream-form T. brucei to stumpy-like forms. (A and B) Bright-field image of Giemsa-stained T. brucei either untreated (A) or treated with 20 μM 8-pCPT-2′-O-Me-5′-AMP for 48 h (B). (C) Diaphorase staining of T. brucei treated with 20 μM 8-pCPT-2′-O-Me-5′-AMP for 48 h observed with phase-contrast microscopy. (D) Survival and proliferation over time (hours) of untreated T. brucei (squares) or T. brucei pretreated for 48 h with 8-pCPT-cAMP (triangles), 8-pCPT-2′-O-Me-cAMP (inverted triangles), or 8-pCPT-2′-O-Me-5′-AMP (diamonds) in SDM-79 medium containing 3 mM citrate/cis-aconitate at 27°C. (E) Expression of procyclin during in vitro differentiation of 8-pCPT-2′-O-Me-5′-AMP-treated T. brucei (48-h treatment), detected by using an antiprocyclin antibody (49). PC, procyclic T. brucei; BF, untreated bloodstream-form T. brucei. A total of 1 × 10⁶ cells were loaded in each lane. Data shown are representative of three separate experiments.