Abstract
Trypanosoma brucei (TB) cultured in rat blood, bovine serum, or lipid-depleted serum generated distinct differences in cholesterol availability. Whereas cell proliferation of the parasite was relatively unaffected by cholesterol availability, the ratios of cellular ergostenols to cholesterol varied from close to unity to 3 orders of magnitude different with cholesterol as the major sterol (>99%) of bloodstream form cells. In the procyclic form cultured with lipid-depleted serum, 15 sterols at 52 fg/cell were identified by GC-MS. The structures of these sterols reveal a nonconventional ergosterol pathway consistent with the novel product diversity catalyzed by the recently cloned sterol methyltransferase (SMT). A potent transition state analog of the TB SMT C24 alkylation reaction, 25-azalanosterol (25-AL; inhibition constant Ki = 39 nM), was found to inhibit the growth of the procyclic and bloodstream forms at an IC(50) of approximately 1 microM. This previously unrecognized catalyst-specific inhibition of cell growth was unmasked further using the 25-AL-treated procyclic form, which, compared with control cultures, caused a change in cellular sterol content from ergostenols to cholesterol. However, growth of the bloodstream form disrupted by 25-AL was not rescued by cholesterol absorption from the host, suggesting an essential role for ergosterol (24-methyl sterol) in cell proliferation and that the SMT can be a new enzyme target for drug design.
Highlights
Trypanosoma brucei (TB) cultured in rat blood, bovine serum, or lipid-depleted serum generated distinct differences in cholesterol availability
A further inconsistency is that the ergosterol pathway in the procyclic form of TB is considered to be similar to that of T. cruzi and other protozoa [6, 7], the recent enzymegenerated product set of the cloned C24 sterol methyltransferase (SMT) [10] indicates that TB should possess an ergosterol pathway that is atypical for protozoa and similar in many ways to the side chain pathway synthesized by the nonphotosynthetic alga Prototheca wickerhamii [14]
Consistent with the findings of others [6, 12], that TB cells can accumulate vast amounts of human cholesterol concurrent with a downregulation of the native ergosterol pathway, is intriguing because it implies that the protozoan has developed some degree of sterol auxotrophy and readily will absorb a variety of exogenous sterols to permit sterols to play a dual function in trypanosomes: one structural, which can be satisfied by exogenous sterol, and the other metabolic, for which there are more strict requirements, satisfied only by biosynthesis
Summary
Trypanosoma brucei (TB) cultured in rat blood, bovine serum, or lipid-depleted serum generated distinct differences in cholesterol availability. A further inconsistency is that the ergosterol pathway in the procyclic form of TB is considered to be similar to that of T. cruzi and other protozoa [6, 7], the recent enzymegenerated product set of the cloned C24 SMT [10] indicates that TB should possess an ergosterol pathway that is atypical for protozoa and similar in many ways to the side chain pathway synthesized by the nonphotosynthetic alga Prototheca wickerhamii [14] These anomalies, together with our interest in designing taxa-specific, catalyst-based drugs tailored to interfere with SMT activity [14,15,16], led us to reinvestigate the sterol composition of TB cultured under various conditions of cholesterol availability and in the presence of an inhibitor discovered by us to block catalysis of the cloned TB SMT [10]. As reported here, a transition state analog of the sterol C24 methylation step in ergosterol synthesis is an effective growth inhibitor against both the procyclic and bloodstream forms
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