PhD Student, University of KwaZulu Natal
Expression and characterisation of heavy metal binding proteins from African trypanosomes.
We are interested in the pathway of transport of heavy metals within the trypanosomes. This pathway is important for two paradoxical reasons: 1. Heavy metals are essential in trypanosomes 2. In their free form, these heavy metals are very toxic to the cell. Therefore, cells must constantly deal with the tight regulation of free levels of heavy metals. My research involves identification of proteins encoded in the genomes of African trypanosomes involved in the binding of heavy metals. Using molecular biology and biochemical techniques these genes were “isolated” and translated into proteins (termed recombinant expression) for characterisation. Using immunological techniques, the presence of these proteins was validated in the parasites cultured in our lab. This lays a foundation for the investigation of possible roles of these proteins in the survival of African trypanosomes and for use as targets.
Abstract: Gambian sleeping sickness or HAT (human African trypanosomiasis) is a neglected tropical disease caused by Trypanosoma brucei gambiense transmitted by riverine species of tsetse. A global programme aims to eliminate the disease as a public health problem by 2020 and stop transmission by 2030. In the South of Chad, the Mandoul area is a persistent focus of Gambian sleeping sickness where around 100 HAT cases were still diagnosed and treated annually until 2013. Pre-2014, control of HAT relied solely on case detection and treatment, which lead to a gradual decrease in the number of cases of HAT due to annual screening of the population.Because of the persistence of transmission and detection of new cases, we assessed whether the addition of vector control to case detection and treatment could further reduce transmission and consequently, reduce annual incidence of HAT in Mandoul. In particular, we investigated the impact of deploying 'tiny targets' which attract and kill tsetse. Before tsetse control commenced, a census of the human population was conducted and their settlements mapped. A pre-intervention survey of tsetse distribution and abundance was implemented in November 2013 and 2600 targets were deployed in the riverine habitats of tsetse in early 2014, 2015 and 2016. Impact on tsetse and on the incidence of sleeping sickness was assessed through nine tsetse monitoring surveys and four medical surveys of the human population in 2014 and 2015. Mathematical modelling was used to assess the relative impact of tsetse control on incidence compared to active and passive screening.The census indicated that a population of 38674 inhabitants lived in the vicinity of the Mandoul focus. Within this focus in November 2013, the vector is Glossina fuscipes fuscipes and the mean catch of tsetse from traps was 0.7 flies/trap/day (range, 0-26). The catch of tsetse from 44 sentinel biconical traps declined after target deployment with only five tsetse being caught in nine surveys giving a mean catch of 0.005 tsetse/trap/day. Modelling indicates that 70.4% (95% CI: 51-95%) of the reduction in reported cases between 2013 and 2015 can be attributed to vector control with the rest due to medical intervention. Similarly tiny targets are estimated to have reduced new infections dramatically with 62.8% (95% CI: 59-66%) of the reduction due to tsetse control, and 8.5% (95% 8-9%) to enhanced passive detection. Model predictions anticipate that elimination as a public health problem could be achieved by 2018 in this focus if vector control and screening continue at the present level and, furthermore, there may have been virtually no transmission since 2015.This work shows that tiny targets reduced the numbers of tsetse in this focus in Chad, which may have interrupted transmission and the combination of tsetse control to medical detection and treatment has played a major role in reducing in HAT incidence in 2014 and 2015.
Pub.: 28 Jul '17, Pinned: 09 Nov '17
Abstract: Trypanosoma brucei is transmitted between mammalian hosts by the tsetse fly. In the mammal, they are exclusively extracellular, continuously replicating within the bloodstream. During this stage, the mitochondrion lacks a functional electron transport chain (ETC). Successful transition to the fly, requires activation of the ETC and ATP synthesis via oxidative phosphorylation. This life cycle leads to a major problem: in the bloodstream, the mitochondrial genes are not under selection and are subject to genetic drift that endangers their integrity. Exacerbating this, T. brucei undergoes repeated population bottlenecks as they evade the host immune system that would create additional forces of genetic drift. These parasites possess several unique genetic features, including RNA editing of mitochondrial transcripts. RNA editing creates open reading frames by the guided insertion and deletion of U-residues within the mRNA. A major question in the field has been why this metabolically expensive system of RNA editing would evolve and persist. Here, we show that many of the edited mRNAs can alter the choice of start codon and the open reading frame by alternative editing of the 5' end. Analyses of mutational bias indicate that six of the mitochondrial genes may be dual-coding and that RNA editing allows access to both reading frames. We hypothesize that dual-coding genes can protect genetic information by essentially hiding a non-selected gene within one that remains under selection. Thus, the complex RNA editing system found in the mitochondria of trypanosomes provides a unique molecular strategy to combat genetic drift in non-selective conditions.
Pub.: 11 Oct '17, Pinned: 09 Nov '17
Abstract: Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. The mRNAs are differentially edited in bloodstream form (BF) and procyclic form (PF) life cycle stages, and this correlates with the differential utilization of glycolysis and oxidative phosphorylation between the stages. The mechanism that controls this differential editing is unknown. Editing is catalyzed by multiprotein ∼20S editosomes that contain endonuclease, 3'-terminal uridylyltransferase, exonuclease, and ligase activities. These editosomes also contain KREPB5 and KREPA3 proteins, which have no functional catalytic motifs, but they are essential for parasite viability, editing, and editosome integrity in BF cells. We show here that repression of KREPB5 or KREPA3 is also lethal in PF, but the effects on editosome structure differ from those in BF. In addition, we found that point mutations in KREPB5 or KREPA3 differentially affect cell growth, editosome integrity, and RNA editing between BF and PF stages. These results indicate that the functions of KREPB5 and KREPA3 editosome proteins are adjusted between the life cycle stages. This implies that these proteins are involved in the processes that control differential editing and that the 20S editosomes differ between the life cycle stages.
Pub.: 26 Aug '15, Pinned: 09 Nov '17
Abstract: The mitochondrial outer membrane (OM) contains single and multiple membrane-spanning proteins which need to contain signals that ensure correct targeting and insertion into the OM. The biogenesis of such proteins has so far essentially only been studied in yeast and related organisms. Here we show that POMP10, an OM protein of the early diverging protozoan Trypanosoma brucei, is signal-anchored. Transgenic cells expressing variants of POMP10 fused to GFP demonstrate that the N-terminal membrane-spanning domain flanked by a few positively charged or neutral residues is both necessary and sufficient for mitochondrial targeting. Carbonate extraction experiments indicate that, while the presence of neutral instead of positively charged residues did not interfere with POMP10 localization, it weakened its interaction with the OM. Expression of GFP-tagged POMP10 in inducible RNAi cell lines shows that its mitochondrial localization depends on pATOM36 but does neither require Sam50 nor ATOM40, the trypanosomal analogue of the Tom40 import pore. pATOM36 is a kinetoplastid-specific OM protein that has previously been implicated in the assembly of OM proteins and in mitochondrial DNA inheritance. In summary, our results show that while the features of the targeting signal in signal-anchored proteins are widely conserved, the protein machinery that mediates their biogenesis is not.
Pub.: 18 Jan '17, Pinned: 09 Nov '17
Abstract: The mitochondrial contact site and cristae organizing system (MICOS) is a recently discovered protein complex that is crucial for establishing and maintaining the proper inner membrane architecture and contacts with the outer membrane of mitochondria. The ways in which the MICOS complex is assembled and its integrity is regulated remain elusive. Here, we report a direct link between Cox17, a protein involved in the assembly of cytochrome c oxidase, and the MICOS complex. Cox17 interacts with Mic60, thereby modulating MICOS complex integrity. This interaction does not involve Sco1, a partner of Cox17 in transferring copper ions to cytochrome c oxidase. However, the Cox17-MICOS interaction is regulated by copper ions. We propose that Cox17 is a newly identified factor involved in maintaining the architecture of the MICOS complex.
Pub.: 29 Apr '15, Pinned: 09 Nov '17
Abstract: Copper is an essential metal ion for embryonic development, iron acquisition, cardiac function, neuropeptide biogenesis and other critical physiological processes. Ctr1 is a high affinity Cu+ transporter on the plasma membrane and endosomes that exists as a full length protein and a truncated form (tCtr1) lacking the methionine and histidine rich metal binding ecto-domain and which exhibits reduced Cu+ transport activity. Here we identify the cathepsin L/B endo-lysosomal proteases functioning in a direct and rate-limiting step in Ctr1 ecto-domain cleavage. Cells and mice lacking cathepsin L accumulate full length Ctr1 and hyper-accumulate Cu. As Ctr1 also transports the chemotherapeutic drug cisplatin via direct binding to the ecto-domain, we demonstrate that the combination of cisplatin with a cathepsin L/B inhibitor enhances cisplatin uptake and cell killing. These studies identify a new processing event, and the key protease that cleaves the Ctr1 metal-binding ecto-domain, that functions to regulate cellular Cu+ and cisplatin acquisition.
Pub.: 03 May '16, Pinned: 09 Nov '17
Abstract: Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease (MD) and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from cytosol into the secretory pathway. Using fibroblasts from MD patients and mouse 3T3-L1 cells with a CRISPR/Cas9 inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation, i.e. elevated copper, and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondria morphology or activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox mis-balance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).
Pub.: 16 May '16, Pinned: 09 Nov '17
Abstract: Metal efflux pumps maintain ion homeostasis in the cell. The functions of the transporters are often supported by chaperone proteins, which scavenge the metal ions from the cytoplasm. Although the copper ion transporter CopA has been known in Escherichia coli, no gene for its chaperone had been identified. We show that the CopA chaperone is expressed in E. coli from the same gene that encodes the transporter. Some ribosomes translating copA undergo programmed frameshifting, terminate translation in the -1 frame, and generate the 70 aa-long polypeptide CopA(Z), which helps cells survive toxic copper concentrations. The high efficiency of frameshifting is achieved by the combined stimulatory action of a "slippery" sequence, an mRNA pseudoknot, and the CopA nascent chain. Similar mRNA elements are not only found in the copA genes of other bacteria but are also present in ATP7B, the human homolog of copA, and direct ribosomal frameshifting in vivo.
Pub.: 21 Jan '17, Pinned: 09 Nov '17
Abstract: Copper plays a dual role as a nutrient and a toxin during bacterial infections. While uropathogenic Escherichia coli (UPEC) strains can use the copper-binding metallophore yersiniabactin (Ybt) to resist copper toxicity, Ybt also converts bioavailable copper to Cu(II)–Ybt in low-copper conditions. Although E. coli have long been considered to lack a copper import pathway, we observed Ybt-mediated copper import in UPEC using canonical Fe(III)–Ybt transport proteins. UPEC removed copper from Cu(II)–Ybt with subsequent re-export of metal-free Ybt to the extracellular space. Copper released through this process became available to an E. coli cuproenzyme (the amine oxidase TynA), linking this import pathway to a nutrient acquisition function. Ybt-expressing E. coli thus engage in nutritional passivation, a strategy of minimizing a metal ion's toxicity while preserving its nutritional availability. Copper acquisition through this process may contribute to the marked virulence defect of Ybt-transport-deficient UPEC.
Pub.: 24 Jul '17, Pinned: 09 Nov '17
Abstract: Bacterial copper (Cu(+)) homeostasis enables both precise metallation of diverse cuproproteins and control of variable metal levels. To this end, protein networks mobilize Cu(+) to cellular targets with remarkable specificity. However, the understanding of these processes is rather fragmented. Here, we use genome-wide transcriptomic analysis by RNA-Seq to characterize the response of Pseudomonas aeruginosa to external 0.5 mm CuSO4, a condition that did not generate pleiotropic effects. Pre-steady-state (5-min) and steady-state (2-h) Cu(+) fluxes resulted in distinct transcriptome landscapes. Cells quickly responded to Cu(2+) stress by slowing down metabolism. This was restored once steady state was reached. Specific Cu(+) homeostasis genes were strongly regulated in both conditions. Our system-wide analysis revealed induction of three Cu(+) efflux systems (a P1B-ATPase, a porin, and a resistance-nodulation-division (RND) system) and of a putative Cu(+)-binding periplasmic chaperone and the unusual presence of two cytoplasmic CopZ proteins. Both CopZ chaperones could bind Cu(+) with high affinity. Importantly, novel transmembrane transporters probably mediating Cu(+) influx were among those largely repressed upon Cu(+) stress. Compartmental Cu(+) levels appear independently controlled; the cytoplasmic Cu(+) sensor CueR controls cytoplasmic chaperones and plasma membrane transporters, whereas CopR/S responds to periplasmic Cu(+) Analysis of ΔcopR and ΔcueR mutant strains revealed a CopR regulon composed of genes involved in periplasmic Cu(+) homeostasis and its putative DNA recognition sequence. In conclusion, our study establishes a system-wide model of a network of sensors/regulators, soluble chaperones, and influx/efflux transporters that control the Cu(+) levels in P. aeruginosa compartments.
Pub.: 02 Aug '17, Pinned: 09 Nov '17
Abstract: The human transporter ATP7B delivers copper to the biosynthetic pathways and maintains copper homeostasis in the liver. Mutations in ATP7B cause the potentially fatal hepato-neurological disorder Wilson disease. The activity and intracellular localization of ATP7B are regulated by copper, but the molecular mechanism of this regulation is largely unknown. We show that the copper chaperone Atox1, which delivers copper to ATP7B, and the group of the first three metal binding domains (MBD1-3) are central to the activity regulation of ATP7B. Atox1-Cu binding to ATP7B changes domain dynamics and interactions within the MBD1-3 group, and activates ATP hydrolysis. To understand the mechanism linking Atox1-MBD interactions and enzyme activity, we have determined the MBD1-3 conformational space using SAXS, and identified changes in MBD dynamics caused by apo-Atox1 and Atox1-Cu by solution NMR. The results show that copper transfer from Atox1 decreases domain interactions within the MBD1-3 group and increases the mobility of the individual domains. The N-terminal segment of MBD1-3 was found to interact with the nucleotide-binding domain of ATP7B, thus physically coupling the domains involved in copper binding and those involved in ATP hydrolysis. Taken together, the data suggest a regulatory mechanism, in which Atox1-mediated Cu-transfer activates ATP7B by releasing inhibitory constraints through increased freedom of MBD1-3 motions.
Pub.: 14 Sep '17, Pinned: 09 Nov '17