Novel aspects of iron homeostasis in pathogenic bloodstream form Trypanosoma brucei

Abstract

Iron is an essential regulatory signal for virulence factors in many pathogens. Mammals and bloodstream form (BSF) Trypanosoma brucei obtain iron by receptor-mediated endocytosis of transferrin bound to receptors (TfR) but the mechanisms by which T. brucei subsequently handles iron remains enigmatic. Here, we analyse the transcriptome of T. brucei cultured in iron-rich and iron-poor conditions. We show that adaptation to iron-deprivation induces upregulation of TfR, a cohort of parasite-specific genes (ESAG3, PAGS), genes involved in glucose uptake and glycolysis (THT1 and hexokinase), endocytosis (Phosphatidic Acid Phosphatase, PAP2), and most notably a divergent RNA binding protein RBP5, indicative of a non-canonical mechanism for regulating intracellular iron levels. We show that cells depleted of TfR by RNA silencing import free iron as a compensatory survival strategy. The TfR and RBP5 iron response are reversible by genetic complementation, the response kinetics are similar, but the regulatory mechanisms are distinct. Increased TfR protein is due to increased mRNA. Increased RBP5 expression, however, occurs by a post-transcriptional feedback mechanism whereby RBP5 interacts with its own, and with PAP2 mRNAs. Further observations suggest that increased RBP5 expression in iron-deprived cells has a maximum threshold as ectopic overexpression above this threshold disrupts normal cell cycle progression resulting in an accumulation of anucleate cells and cells in G2/M phase. This phenotype is not observed with overexpression of RPB5 containing a point mutation (F61A) in its single RNA Recognition Motif. Our experiments shed new light on how T. brucei BSFs reorganise their transcriptome to deal with iron stress revealing the first iron responsive RNA binding protein that is co-regulated with TfR, is important for cell viability and iron homeostasis; two essential processes for successful proliferation.

Stress conditions promote Leishmania hybridization in vitro marked by expression of the ancestral gamete fusogen HAP2 as revealed by single-cell RNA-seq

“…From the 708 genes upregulated post-irradiation in L-cluster2 and 296 genes in M-cluster1, 169 are common between the two strains ( Figure 4B , Supplementary file 4 ). This list includes repressor of differentiation kinase 2 ( RDK2 ; LmjF.31.2960) and RNA-binding protein 5 ( RBP5 ; LmjF.09.0060), both of which are involved in Trypanosoma brucei proliferation ( Gilabert Carbajo et al, 2021 ; Jones et al, 2014 ). Among the top 20 most upregulated genes in either L-cluster2 or M-cluster1 are seven genes common to both lists: serine/threonine-protein kinase NEK15 (LmjF.26.2570), adenylate-cyclase ACP2 (LmjF.10.0540), three putative surface antigen proteins (LmjF.05.1215; LmjF.12.1090 and LmjF.35.0550), one gene of unknown function (LmjF.26.2680), and a putative leucine-rich repeat (LRR) protein annotated as a pseudogene on TriTrypDB (LmjF.31.1440) ( Figure 4B ).…”

The vacuolar iron transporter mediates iron detoxification inToxoplasma gondii

“…Our data reveal that VIT levels drop transcriptionally upon iron excess, and at the protein level both upon iron depletion and excess. Transcriptional and post-transcriptional control of iron uptake and storage have been examined in detail in bacteria, yeast, plants and mammalian cells (Andrews et al, 2003;Gao and Dubos, 2021;Li et al, 2008;Philpott et al, 2012;Wang and Pantopoulos, 2011), and was recently also demonstrated in the kinetoplastid parasite Trypansoma brucei (Carbajo et al, 2021). However, iron-mediated regulation has not previously been observed in the Apicomplexa.…”

“…This iron is required for a number of essential cellular processes, including heme biosynthesis (Bergmann et al, 2020;Kloehn et al, 2020), iron sulphur (Fe-S) cluster biogenesis (Aw et al, 2020), and as a cofactor in several important proteins such as catalase (Kwok et al, 2004;Odberg-Ferragut et al, 2000), prolyl hydroxylases (Florimond et al, 2019) and the deoxyribonucleotide synthesis enzyme ribonucleotide reductase. Iron acquisition and use have been studied in various pathogenic parasites such as Trichomonas (Sehgal et al, 2012) and in some detail in kinetoplastids such as Leishmania (Zaidi et al, 2017) and Trypanosoma (Carbajo et al, 2021). However, iron utilisation remains understudied in the apicomplexan parasites.…”

Generation of a bloodstream formTrypanosoma bruceidouble glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin

“…Endocytosis assays-Endocytosis was performed as previously described [20]. Briefly, logphase trypanosome cells were washed in Hepes-buffered saline (HBS; 50 mM Hepes KOH, pH 7.5, 50 mM NaCl, 5 mM KCl, 70 mM glucose) and pre-incubated (10 6 /ml) in serum-free HMI9 with BSA (0.5 mg/ml) for 10 minutes at 37°C.…”