Stephen L. Hajduk

Human High Density Lipoproteins Are Platforms for the Assembly of Multi-component Innate Immune Complexes

Human innate immunity to non-pathogenic species of African trypanosomes is provided by human high density lipoprotein (HDL) particles. Here we show that native human HDLs containing haptoglobin-related protein (Hpr), apolipoprotein L-I (apoL-I) and apolipoprotein A-I (apoA-I) are the principle antimicrobial molecules providing protection from trypanosome infection. Other HDL subclasses containing either apoA-I and apoL-I or apoA-I and Hpr have reduced trypanolytic activity, whereas HDL subclasses lacking apoL-I and Hpr are non-toxic to trypanosomes. Highly purified, lipid-free Hpr and apoL-I were both toxic to Trypanosoma brucei brucei but with specific activities at least 500-fold less than those of native HDLs, suggesting that association of these apolipoproteins within the HDL particle was necessary for optimal cytotoxicity. These studies show that HDLs can serve as platforms for the assembly of multiple synergistic proteins and that these assemblies may play a critical role in the evolution of primate-specific innate immunity to trypanosome infection.

Native mRNA editing complexes from Trypanosoma brucei mitochondria.

The aim of this study was to identify multicomponent complexes involved in kinetoplastid mitochondrial mRNA editing. Mitochondrial extracts from Trypanosoma brucei were fractionated on 10–30% glycerol gradients and assayed for RNAs and activities potentially involved in editing, including pre‐edited mRNA, guide RNA (gRNA), endonuclease, terminal uridylyltransferase (TUTase), RNA ligase and gRNA‐mRNA chimera‐forming activities. These experiments suggest that two distinct editing complexes exist. Complex I (19S) consists of gRNA, TUTase, RNA ligase and chimera‐forming activity. Complex II (35–40S) is composed of gRNA, preedited mRNA, RNA ligase and chimera‐forming activity. These studies provide the first evidence that editing occurs in a multicomponent complex. The possible roles of complex I, complex II and RNA ligase in editing are discussed.

Editing Machines: The Complexities of Trypanosome RNA Editing

The assembly and disassembly of ribonucleoprotein complexes containing substrate precursor mRNAs and guide RNAs is crucial to the initiation and propagation of RNA editing. We discuss here the composition of these complexes and how their assembly may regulate RNA editing.

Developmental regulation of mitochondrial biogenesis inTrypanosoma brucei

The metabolism ofTrypanosoma brucei undergoes a significant change as the parasite differentiates from the mammalian bloodstream form to the form found in the tse-tse fly vector. Because the mitochondria of bloodstream form cells lack cytochromes and several key citric acid cycle enzymes, the metabolism of these cells is mostly limited to glycolysis. The reducing equivalents generated by this process are passed to oxygen by a plantlike alternative oxidase. As cells differentiate to the insect form, they begin to oxidatively metabolize proline. The mitochondria of insect form cells contain functional, cytochromemediated electron transport chains and have complete complements of citric acid cycle enzymes. Although the characterization is far from complete, the nuclear and mitochondrial genes involved in the expression of these mitochondrial functions appear to be developmentally regulated at posttranscriptional and posttranslational levels. This review outlines some of the molecular processes that are associated with the developmental regulation of mitochondrial biogenesis and suggests some possible mechanisms of regulation.

RNA editing in kinetoplastid mitochondria.

RNA editing in the mitochondrion of kinetoplastid protozoa results in the posttranscriptional addition and deletion of uridine residues in mRNAs. Editing of mRNAs can lead to the formation of initiation codons for mitochondrial translation, the correction of frame‐shifted genes at the RNA level, and in extensively edited mRNAs, the formation of complete reading frames. Kinetoplastid RNA editing requires that genetic information from two or more separately transcribed genes be brought together to form the mature, edited mRNA. The information necessary for the proper insertion or deletion of uridines in the mRNA is present in small mitochondrial transcripts termed guide RNAs (gRNAs). Editing of mRNAs appears to be associated with a high molecular weight complex, called the editosome, containing specific gRNAs, unedited mRNAs, and proteins. Editing is likely a two‐step process involving first the breakage of a phosphodiester bond at the editing site and formation of a chimeric molecule with a gRNA covalently joined to the 5′ end of the 3′ portion of an mRNA. The chimera is resolved by the rejoining of the 5′ end of the mRNA to the 3′ portion of the mRNA with the addition or deletion of a uridine at the junction point. Two models are proposed for the biochemical mechanism of RNA editing. The first is an enzymatic cascade of cleavage and ligation while the other supports successive rounds of transesterification. The obvious functional necessity for editing in kinetoplastid mitochondria is the formation of translatable mRNAs. Far less clear is the evolutionary origin of editing and the role editing plays in regulating mitochondrial gene expression.— Hajduk, S. L., Harris, M. E., and Pollard, V. W. RNA editing in kinetoplastid mitochondria. FASEB J. 7: 54‐63; 1993.

Mechanism of Trypanosoma brucei gambiense (group 1) resistance to human trypanosome lytic factor

Human innate immunity against most African trypanosomes, including Trypanosoma brucei brucei, is mediated by a minor subclass of toxic serum HDL, called trypanosome lytic factor-1 (TLF-1). This HDL contains two primate specific proteins, apolipoprotein L-1 and haptoglobin (Hp)-related protein, as well as apolipoprotein A-1. These assembled proteins provide a powerful defense against trypanosome infection. Trypanosoma brucei rhodesiense causes human African sleeping sickness because it has evolved an inhibitor of TLF-1, serum resistance-associated (SRA) protein. Trypanosoma brucei gambiense lacks the SRA gene, yet it infects humans. As transfection of T. b. gambiense (group 1) is not possible, we initially used in vitro-selected TLF-1–resistant T. b. brucei to examine SRA-independent mechanisms of TLF-1 resistance. Here we show that TLF-1 resistance in T. b. brucei is caused by reduced expression of the Hp/Hb receptor gene (TbbHpHbR). Importantly, T. b. gambiense (group 1) also showed a marked reduction in uptake of TLF-1 and a corresponding decrease in expression of T. b. gambiense Hp/Hb receptor (TbgHpHbR). Ectopic expression of TbbHpHbR in TLF-1–resistant T. b. brucei rescued TLF-1 uptake, demonstrating that decreased TbbHpHbR expression conferred TLF-1 resistance. Ectopic expression of TbgHpHbR in TLF-1–resistant T. b. brucei failed to rescue TLF-1 killing, suggesting that coding sequence changes altered Hp/Hb receptor binding affinity for TLF-1. We propose that the combination of coding sequence mutations and decreased expression of TbgHpHbR directly contribute to parasite evasion of human innate immunity and infectivity of group 1 T. b. gambiense.

Extracellular Vesicles from Trypanosoma brucei Mediate Virulence Factor Transfer and Cause Host Anemia

Intercellular communication between parasites and with host cells provides mechanisms for parasite development, immune evasion, and disease pathology. Bloodstream African trypanosomes produce membranous nanotubes that originate from the flagellar membrane and disassociate into free extracellular vesicles (EVs). Trypanosome EVs contain several flagellar proteins that contribute to virulence, and Trypanosoma brucei rhodesiense EVs contain the serum resistance-associated protein (SRA) necessary for human infectivity. T. b. rhodesiense EVs transfer SRA to non-human infectious trypanosomes, allowing evasion of human innate immunity. Trypanosome EVs can also fuse with mammalian erythrocytes, resulting in rapid erythrocyte clearance and anemia. These data indicate that trypanosome EVs are organelles mediating non-hereditary virulence factor transfer and causing host erythrocyte remodeling, inducing anemia.

Alternative editing of cytochrome c oxidase III mRNA in trypanosome mitochondria generates protein diversity

Trypanosomes use RNA editing to produce most functional mitochondrial messenger RNA. Precise insertion and deletion of hundreds of uridines is necessary to make full‐length cytochrome c oxidase III (COXIII) mRNA. We show that COXIII mRNA can be alternatively edited by a mechanism using an alternative guide RNA to make a stable mRNA. This alternatively edited mRNA is translated to produce a unique protein that fractionates with mitochondrial membranes and colocalizes with mitochondrial proteins in situ. Alternative RNA editing represents a previously unknown mechanism generating protein diversity and, as such, represents an important function for RNA editing.

The LAMP‐like protein p67 plays an essential role in the lysosome of African trypanosomes

RNAi knockdown was employed to study the function of p67, a lysosome‐associated membrane protein (LAMP)‐like type I transmembrane lysosomal glycoprotein in African trypanosomes. Conditional induction of p67 dsRNA resulted in specific ∼90% reductions in de novo p67 synthesis in both mammalian bloodstream and procyclic insect‐stage parasites. Bloodstream cell growth was severely retarded with extensive death after > 24 h of induction. Biosynthetic trafficking of residual p67, and of the soluble lysosomal protease trypanopain, were unimpaired. Endocytosis of tomato lectin, a surrogate receptor‐mediated cargo, was only mildly impaired (∼20%), but proper lysosomal targeting was unaffected. p67 ablation had dramatic effects on lysosomal morphology with gross enlargement (four‐ to fivefold) and internal membrane profiles reminiscent of autophagic vacuoles. Ablation of p67 expression rendered bloodstream trypanosomes refractory to lysis by human trypanolytic factor (TLF), a lysosomally activated host innate immune mediator. Similar effects on lysosomal morphology and TLF sensitivity were also obtained by two pharmacological agents that neutralize lysosomal pH – chloroquine and bafilomycin A1. Surprisingly, however, lysosomal pH was not affected in ablated cells suggesting that other physiological alterations must account for increased resistance to TLF. These results indicate p67 plays an essential role in maintenance of normal lysosomal structure and physiology in bloodstream‐stage African trypanosomes.

A Trypanosome Mitochondrial RNA Polymerase Is Required for Transcription and Replication

Understanding mitochondrial transcription is a requisite first step toward understanding the regulation of mitochondrial gene expression in kinetoplastids. Here we report the identification and functional characterization of a mitochondrial RNA polymerase (mtRNAP) from Trypanosoma brucei, the firsttrans-acting factor involved in kinetoplast mitochondrial transcription to be identified. Using sequences conserved among the catalytic domains of the single-subunit mtRNAPs, we were able to obtain a full-length sequence for a candidate mtRNAP from T. brucei. Sequence comparison indicates that it shares homology in its catalytic domain with other single-subunit mtRNAPs, including functionally conserved residues that are identical in all single-subunit RNAPs. We used RNA interference to functionally knock out the gene product to determine whether the candidate gene represents an mtRNAP. As predicted for a mitochondrial specific RNA polymerase, reduction of the gene product resulted in a specific decrease of mitochondrial versus nuclear transcripts. Additionally, similar to the mtRNAP of other organisms, the mtRNAP characterized here is involved in replication of the mitochondrial genome. Thus, based on sequence comparison and functional studies, we have cloned an mtRNAP from trypanosomes.