ABSTRACT
Transfer of bloodstream-form Trypanosoma brucei variant 221a from calf serum to dog serum-based medium induces acute iron starvation, as the transferrin receptor (Tf-R) of variant 221a binds dog Tf poorly. We show here that transfer to dog serum induces a 3-5-fold increase in Tf-R mRNA and protein within one doubling time (8 h). Because iron stores are still high 8 h after transfer, we infer that the signal for Tf-R overproduction is the decreased availability of cytosolic iron when cellular iron import drops. Up to 30% of the extra Tf-R spills out of the flagellar pocket onto the pellicular surface. Because the 5-fold increase in Tf-R is accompanied by a 5-fold increase in bovine Tf uptake, the up-regulation of Tf-R levels in response to Tf starvation helps the trypanosome to compete for limiting amounts of Tf. We noted that Tf-R levels also vary in calf serum medium. Cells in dense cultures contain up to 5-fold more Tf-R mRNA and protein than in dilute cultures. Only one-tenth of the extra Tf-R reaches the pellicular surface. The increase cannot be explained by a lack of Tf or to cell density sensing but is due to pericellular hypoxia. Our results show that bloodstream-form trypanosomes can regulate the expression of the two Tf-R subunit genes and the localization of their gene products in a flexible manner. This flexibility is made possible by the promoter-proximal position of the two genes in the variant surface glycoprotein expression site.
Subject(s)
Receptors, Transferrin/biosynthesis , Trypanosoma brucei brucei/metabolism , Animals , Binding Sites , Blotting, Northern , Cattle , Culture Media/metabolism , Cytosol/metabolism , Dogs , Dose-Response Relationship, Drug , Electron Probe Microanalysis , Endocytosis , Flagella/metabolism , Glycosylation , Hypoxia , Iron/metabolism , Mice , Microscopy, Fluorescence , Protein Binding , RNA, Messenger/metabolism , Receptors, Transferrin/metabolism , Time Factors , Up-RegulationABSTRACT
Trypanosoma brucei escapes destruction by the host immune system by regularly replacing its Variant Surface Glycoprotein (VSG) coat. The VSG is expressed in a VSG expression site, together with expression site associated gene (ESAG) 6 and 7, encoding the heterodimeric transferrin receptor (Tf-R). There are around 20 VSG expression sites, and trypanosomes can change the site that is active. Since ESAG6 and 7 in different expression sites differ somewhat in sequence, expression site switching results in the production of a slightly different Tf-R. We have studied the physiological relevance of Tf-R variation for the survival of T. brucei in mammalian sera. Trypanosomes with an active 221 expression site, encoding a Tf-R with a very low affinity for canine Tf (Kd>1 microM), were cultured in canine serum based medium. This resulted in selection of trypanosomes that had switched to the VO2, the 223 or the bR-2 expression site, each encoding a Tf-R with higher affinity for canine Tf than the 221 site Tf-R. Adding bovine Tf to the medium could prevent the switch, indicating that the low uptake of Tf provided the selection against 221 trypanosomes. Horse serum based medium also induced switching to the VO2 expression site, but this was not prevented by bovine Tf. In the presence of physiological concentrations of anti-Tf-R antibody, only a high-affinity Tf allowed the growth of 221 Tf-R expressing trypanosomes. Our results suggest that a high-affinity Tf-R not only ensures efficient Tf uptake, but is also required to allow sufficient iron uptake by the trypanosome in the presence of anti-Tf-R antibodies.
