PhD student, La Trobe University
Study of the molecular interactions between human malarial parasite and its host red blood cell
Malaria is a major health burden worldwide leading to more than half a million deaths every year. It occurs in the tropical regions of the globe and half of the world population is at risk of contracting the disease. In many instances, death can be prevented if symptoms are detected early. However, Plasmodium has developed resistance against all existing treatments, including to the last line treatment recommended by the World Health Organisation. In addition, the number of novel compounds currently in development and in clinical trial is unlikely to meet the need for treatment over the next decade. Identifying novel targets for the development of new anti-malarial compounds is therefore an urgent health priority area.
Malaria is caused by the unicellular parasite Plasmodium and its transmitted to humans by an Anopheles mosquito. During its complex life cycle, Plasmodium develops inside human red blood cells causing all symptoms of disease, such as fever, anemia, coma and ultimately death.
Our laboratory aims to identify essential parasite and host cell molecules that can be targeted with novel anti-malarial drugs. My project specifically aims to study and target human proteins that the parasite requires to survive. Indeed, targeting host proteins is less likely to lead to drug resistance.
Using various methods, we have identified a few red blood cell proteins that the parasite appears to use for its own survival. Confirmation of these results requires genetically modified host cells - in this case red blood cells that lack our candidate proteins. However, red blood cells do not have a nucleus, nor DNA. So direct genetic manipulation is not possible. A way around this problem is to use stem cells. Stem cells can be induced in vitro to become any human cell type, including red blood cells . The aim of my PhD is to genetically modify stem cells (suppressing a specific candidate gene for example), mature them into red blood cells (that will therefore lack the protein we want to test) and infect these modified red blood cells with Plasmodium. This project will unravel interactions between the malaria parasite and its host red blood cell, as well as discover novel human drug targets. If such targets have corresponding available drugs (for instance drugs initially developed for cancer treatment) we will test those compounds as anti-malarials.
Abstract: Recent advances in genome editing with programmable nucleases have opened up new avenues for multiple applications, from basic research to clinical therapy. The ease of use of the technology-and particularly clustered regularly interspaced short palindromic repeats (CRISPR)-will allow us to improve our understanding of genomic variation in disease processes via cellular and animal models. Here, we highlight the progress made in correcting gene mutations in monogenic hereditary disorders and discuss various CRISPR-associated applications, such as cancer research, synthetic biology, and gene therapy using induced pluripotent stem cells. The challenges, ethical issues, and future prospects of CRISPR-based systems for human research are also discussed.
Pub.: 05 Jan '17, Pinned: 30 Aug '17
Abstract: Publication date: 5 May 2016 Source:Cell Stem Cell, Volume 18, Issue 5 Author(s): Dirk Hockemeyer, Rudolf Jaenisch It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments—the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology—have fundamentally reshaped our approach to biomedical research, stem cell biology, and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided and set the stage for their success. Teaser Hockemeyer and Jaenisch discuss the power and limitations of using human induced pluripotent stem cells and CRISPR/Cas9 technology for understanding complex human disease and developing treatments.
Pub.: 17 Jun '16, Pinned: 30 Aug '17
Abstract: Since 2012, the CRISPR-Cas9 system has been quickly and successfully tested in a broad range of organisms and cells including hematopoietic cells. The application of CRISPR-Cas9 in human hematopoietic cells mainly involves the genes responsible for HIV infection, β-thalassemia and sickle cell disease (SCD). The successful disruption of CCR5 and CXCR4 genes in T cells by CRISPR-Cas9 promotes the prospect of the technology in the functional cure of HIV. More recently, eliminating CCR5 and CXCR4 in induced pluripotent stem cells (iPSCs) derived from patients and targeting the HIV genome have been successfully carried out in several laboratories. The outcome from these approaches bring us closer to the goal of eradicating HIV infection. For hemoglobinopathies the ability to produce iPSC-derived from patients with the correction of hemoglobin (HBB) mutations by CRISPR-Cas9 has been tested in a number of laboratories. These corrected iPSCs also show the potential to differentiate into mature erythrocytes expressing high-level and normal HBB. In light of the initial success of CRESPR-Cas9 in target mutated gene(s) in the iPSCs, a combination of genomic editing and autogenetic stem cell transplantation would be the best strategy for root treatment of the diseases, which could replace traditional allogeneic stem cell transplantation.
Pub.: 02 Oct '16, Pinned: 30 Aug '17
Abstract: Apoptosis can occur in red blood cells (RBC) and seems to be involved in hematologic disorders related to many diseases. In malaria it is known that parasitized RBC (pRBC) is involved in the development of anemia and thrombosis; however, non-parasitized RBC (nRBC) apoptosis could amplify these malaria-associated hematologic events. In fact, in experimental malaria, increased levels of apoptosis were observed in nRBC during lethal Plasmodium yoelii 17XL infection, but in human malaria erythrocytic apoptosis has never been studied. The present study was performed to investigate if nRBC apoptosis also occurs in P. vivax and P. falciparum infections.Apoptosis of nRBC was evaluated in blood samples of P. vivax malaria patients and clinically healthly individuals living in Manaus, Brazil, both ex vivo and after incubation of RBC for 24 h. Additionally, the capacity of plasma from P. vivax or P. falciparum patients was tested for induction of in vitro apoptosis of normal RBC from a clinically healthy individual living in a non-endemic malaria region. Apoptosis was detected by flow cytometry using annexin V staining. In contrast to experimental malaria that significantly increased the levels of apoptotic nRBC both ex-vivo and after 24 h of incubation, no significant alteration on apoptotic nRBC rates was detected in P. vivax infected patients when compared with non-infected control individuals. Similar results were observed when plasma of these P. vivax patients was incubated with normal RBC. Conversely, plasma from P. falciparum-infected subjects induced significant apoptosis of these cells.Apoptosis of normal RBC can be induced by plasma from individuals with P. falciparum (but not with P. vivax) malaria. This finding could reflect the existence of erythrocytic apoptosis during infection that could contribute to the pathogenesis of hematological and vascular complications associated with falciparum malaria.
Pub.: 19 Oct '14, Pinned: 30 Aug '17
Abstract: Recently, we have described that apoptosis-like process of red blood cells (RBC) - eryptosis - in malaria is not restricted to parasitized cells, occurring also in non-parasitized RBC (nRBC). Besides to pathogenic proprieties, apoptosis also participates in the innate defense trough restriction of intracellular pathogens propagation. In the present study, we investigated the capacity of P. falciparum parasites to infect eryptotic RBC. Schizont parasitized RBC concentrated by magnetic separation were cultured with eryptotic RBC obtained by ionomycin treatment and, then, parasite growth was evaluated in Giemsa-stained thin blood smears. While parasites infected and developed normally in control non-eryptotic RBC, cultures performed with eryptotic RBC had a marked decrease in parasitaemia. It was noteworthy a great number of free merozoites in eryptotic RBC cultures, indicating that these cells were not susceptible to invasion. We suggest that although eryptosis could be involved in malaria pathogenesis, it could also acting protectively by controlling parasite propagation.
Pub.: 28 Oct '11, Pinned: 30 Aug '17
Abstract: Intracellular pathogens such as viruses and bacteria subvert all the major cellular functions of their hosts. Targeted host processes include protein synthesis, membrane trafficking, modulation of gene expression, antigen presentation, and apoptosis. In recent years, it has become evident that protozoan pathogens, including members of the phylum Apicomplexa, also hijack their host cell's functions to access nutrients and to escape cellular defenses and immune responses. These obligate intracellular parasites provide superb illustrations of the subversion of host cell processes such as the recruitment and reorganization of host cell compartments without fusion around the parasitophorous vacuole of Toxoplasma gondii; the export of Plasmodium falciparum proteins on the surface of infected erythrocytes; and the induced transformation of the lymphocytes infected by Theileria parva, which leads to clonal extension.
Pub.: 13 Sep '08, Pinned: 30 Aug '17
Abstract: A new flow cytometry method that uses an optimized DNA and RNA staining strategy to monitor the growth and development of the Plasmodium falciparum strain W2mef has been used in a pilot study and has identified Bay 43-9006 1, SU 11274 2, and TMC 125 5 as compounds that exhibit potent (<1 microM) overall and ring stage in vitro antimalarial activity.
Pub.: 12 Aug '09, Pinned: 30 Aug '17
Abstract: Plasmodium vivax cannot be maintained in a continuous culture. To overcome this major obstacle to P. vivax research, we have developed an in vitro method to produce susceptible red blood cell (RBC) precursors from freshly isolated human cord hematopoietic stem cells (HSCs), which were activated with erythropoietin to differentiate into erythroid cells. Differentiation and maturation of erythroid cells were monitored using cell surface markers (CD71, CD36, GPA and Fy6). Duffy(+) reticulocytes appeared after 10 days of erythroid cell culture and exponentially increased to high numbers on days 14-16. Beginning on day 10 these erythroid cells, referred to as growing RBCs (gRBCs), were co-cultured with P. vivax-infected blood directly isolated from patients. Parasite-infected gRBCs were detected by Giemsa staining and a P. vivax-specific immunofluorescence assay in 11 out of 14 P. vivax isolates. These P. vivax cultures were continuously maintained for more than 2 weeks by supplying fresh gRBCs; one was maintained for 85 days before discontinuing the culture. Our results demonstrate that gRBCs derived in vitro from HSCs can provide susceptible Duffy(+) reticulocytes for continuous culture of P. vivax. Of particular interest, we discovered that parasites were able to invade nucleated erythroid cells or erythroblasts that are normally in the bone marrow. The possibility that P. vivax causes erythroblast destruction and hence inflammation in the bone marrow needs to be addressed.
Pub.: 06 Jul '07, Pinned: 29 Aug '17
Abstract: Erythrocyte invasion by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion requires a series of extracellular recognition events between erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. None of the few known receptor-ligand interactions involved are required in all parasite strains, indicating that the parasite is able to access multiple redundant invasion pathways. Here, we show that we have identified a receptor-ligand pair that is essential for erythrocyte invasion in all tested P. falciparum strains. By systematically screening a library of erythrocyte proteins, we have found that the Ok blood group antigen, basigin, is a receptor for PfRh5, a parasite ligand that is essential for blood stage growth. Erythrocyte invasion was potently inhibited by soluble basigin or by basigin knockdown, and invasion could be completely blocked using low concentrations of anti-basigin antibodies; importantly, these effects were observed across all laboratory-adapted and field strains tested. Furthermore, Ok(a-) erythrocytes, which express a basigin variant that has a weaker binding affinity for PfRh5, had reduced invasion efficiencies. Our discovery of a cross-strain dependency on a single extracellular receptor-ligand pair for erythrocyte invasion by P. falciparum provides a focus for new anti-malarial therapies.
Pub.: 15 Nov '11, Pinned: 29 Aug '17
Abstract: Population ageing and increase in cancer incidence may lead to a decreased availability of red blood cell units. Thus, finding an alternative source of red blood cells is a highly relevant challenge. The possibility to reproduce in vitro the human erythropoiesis opens a new era, particularly since the improvement in the culture systems allows to produce erythrocytes from induced-Pluripotent Stem Cells (iPSCs), or CD34(+) Hematopoietic Stem Cells (HSCs). iPSCs have the advantage of in vitro self-renewal, but lead to poor amplification and maturation defects (high persistence of nucleated erythroid precursors). Erythroid differentiation from HSC allows a far better amplification and adult-like hemoglobin synthesis. But the inability of these progenitors to self-renew in vitro remains a limit in their use as a source of stem cells. A major improvement would consist in immortalizing these erythroid progenitors so that they could expand indefinitively. Inducible transgenesis is the first way to achieve this goal. To date, the best immortalized-cell models involve strong oncogenes induction, such as c-Myc, Bcl-xL, and mostly E6/E7 HPV16 viral oncoproteins. However, the quality of terminal differentiation of erythroid progenitors generated by these oncogenes is not optimal yet and the long-term stability of such systems is unknown. Moreover, viral transgenesis and inducible expression of oncogenes raise important problems in term of safety, since the enucleation rate is not 100% and no nucleated cells having replicative capacities should be present in the final product.
Pub.: 25 Jul '17, Pinned: 29 Aug '17