PhD Candidate - Carvalho Lab: Molecular Parasitology.
La Trobe University, Melbourne (Australia)
Targeting our own red blood cells to kill the malaria parasite - a new twist for an old disease
Malaria is a major public health burden worldwide: half of the world population is at risk of contracting the disease. Moreover, malaria parasites are becoming resistant against all existing treatments. This resistance is spreading, and represents a terrifying threat. Considering global warming is likely to expand the habitat of malaria transmission mosquitoes, a rise in malaria infections in the near future is realistic.
To defeat it, know the enemy:
Malaria is caused by the unicellular parasite Plasmodium and is transmitted by mosquitoes. Once inside the body, Plasmodium first infects the liver, without giving rise to any symptoms. Then, it heads to the blood, and grows inside our red blood cells, causing fever, anemia, coma and ultimately death.
Our cells as kamikazes:
Our cells are smart: when they get infected by a microbe, they attempt to clear the infection. To do this they “commit suicide”, or in the scientific jargon, they “activate apoptosis” to kill themselves and the microbes they carry. But microbes are smart too: they usually find ways to stop this suicide from happening. They need their host cell to be alive if they want to survive.
Are red blood cells kamikaze as well?
Red blood cells are very particular cells: they do not have a nucleus (hence, no DNA) nor a mitochondria (the “powerhouse of the cell”), two key components of apoptosis. However, they appear to have a “suicide” of their own called eryptosis. Nothing is known about the molecular events underlying eryptosis. This is exactly what I would like to discover, as well as if Plasmodium interferes with this process, and if so, how. If we could help our natural defense mechanism and tip the balance towards suicide of infected cells, we could eliminate malaria.
But… Why would you target your own cells?
It does seem dangerous to do such a thing. But think about this: parasites can easily develop resistance to drugs that target their own molecules. However, they cannot modify the host cell! Therefore, targeting host proteins is less likely to lead to drug resistance.
I aim to 1) understand red blood cell molecular pathways, in particular those that might decide the life or death of the cell 2) if and how Plasmodium interacts with these pathways 3) identify interesting targets for new antimalarial drugs; drugs that, hopefully, won’t lose their efficiency over the years due to resistance.
Abstract: Suicidal erythrocyte death or eryptosis contributes to or even accounts for anemia in a wide variety of clinical conditions, such as iron deficiency, dehydration, hyperphosphatemia, vitamin D excess, chronic kidney disease (CKD), hemolytic-uremic syndrome, diabetes, hepatic failure, malignancy, arteriitis, sepsis, fever, malaria, sickle-cell disease, beta-thalassemia, Hb-C and G6PD-deficiency, Wilsons disease, as well as advanced age. Moreover, eryptosis is triggered by a myriad of xenobiotics and endogenous substances including cytotoxic drugs and uremic toxins. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the erythrocyte surface. Triggers of eryptosis include oxidative stress, hyperosmotic shock, and energy depletion. Signalling involved in the regulation of eryptosis includes Ca2+ entry, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, cyclin-dependent kinase 4, AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and ill-defined tyrosine kinases. Inhibitors of eryptosis may prevent anaemia in clinical conditions associated with enhanced eryptosis and stimulators of eryptosis may favourably influence the clinical course of malaria. Additional experimentation is required to uncover further clinical conditions with enhanced eryptosis, as well as further signalling pathways, further stimulators, and further inhibitors of eryptosis. Thus, a detailed description of the methods employed in the analysis of eryptosis may help those, who enter this exciting research area. The present synopsis describes the experimental procedures required for the analysis of phosphatidylserine exposure at the cell surface with annexin-V, cell volume with forward scatter, cytosolic Ca2+ activity ([Ca2+]i) with Fluo3, oxidative stress with 2',7'-dichlorodihydrofuorescein diacetate (DCFDA), glutathione (GSH) with mercury orange 1(4-chloromercuryphenyl-azo-2-naphthol), lipid peroxidation with BODIPY 581/591 C11 fluorescence, and ceramide abundance with specific antibodies. The contribution of kinases and caspases is defined with the use of the respective inhibitors. It is hoped that the present detailed description of materials and methods required for the analysis of eryptosis encourages further scientists to enter this highly relevant research area.
Pub.: 19 Sep '17, Pinned: 01 Oct '17
Abstract: The 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase inhibitor simvastatin has been shown to trigger apoptosis of several cell types. The substance has thus been proposed as an additional treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the extracellular face of the erythrocyte cell membrane. Signaling contributing to stimulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), induction of oxidative stress, increase of ceramide abundance, and activation of SB203580-sensitive p38 kinase. The present study explored, whether simvastatin induces eryptosis and aimed to shed light on cellular mechanisms involved.Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant.A 48 h exposure of human erythrocytes to simvastatin (1 µg/ml) significantly decreased the forward scatter, significantly augmented the percentage of annexin-V-binding cells, significantly increased Fluo3-fluorescence, and significantly enhanced DCFDA fluorescence. Simvastatin tended to increase ceramide abundance, an effect, however, escaping statistical significance. The effect of simvastatin on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of SB203580 (2 µM).Simvastatin stimulates eryptosis, an effect at least in part due to Ca2+ entry, oxidative stress, and p38 kinase.
Pub.: 21 Sep '17, Pinned: 01 Oct '17
Abstract: The anaplastic lymphoma kinase (ALK) inhibitor ASP3026 is in clinical development for the treatment of ALK expressing non-small cell lung carcinoma (NSCLC). ASP3026 is in part effective by inducing apoptosis of tumor cells. Erythrocytes lack mitochondria and nuclei, key organelles in the execution of apoptosis, but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Eryptosis is triggered by cell stress, such as energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether ASP3026 impacts on eryptosis.Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ loading with Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of ASP3026 (1-4 µg/ml). Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, and cell volume from forward scatter.Treatment with ASP3026 alone did not significantly modify annexin-V-binding or forward scatter. Energy depletion, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. ASP3026 significantly blunted the effect of energy depletion and oxidative stress, but not of ionomycin on annexin-V-binding. ASP3026 did not significantly influence the effect of any maneuver on forward scatter.ASP3026 is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and oxidative stress.
Pub.: 21 Sep '17, Pinned: 01 Oct '17
Abstract: Haemolysis and anaemia related to autoimmune haemolytic anaemia (AIHA) of warm type (wAIHA) and of cold type (cAIHA) are believed to be solely due to antibody and/or complement-mediated destruction and clearance of red blood cells (RBCs). There is evidence that RBCs of affected patients may also undergo eryptosis, the suicidal death of RBCs.RBCs from 24 patients with wAIHA, 7 patients with chronic cAIHA, and one patient with AIHA of mixed-type were analysed for exposed phosphatidylserine (PS) by treatment with phycoerythrin (PE)-labelled Annexin V and cell-associated fluorescence was measured using a MACSQuant flow cytometer.PS exposing RBCs were detected in 7 of 13 patients with clinically significant wAIHA. Haemolysis was mostly related to IgM or IgA autoantibodies (aab) in those patients. In contrast, PS exposure in 11 wAIHA patients in complete remission was comparable to that in healthy blood donors. All patients with chronic cAIHA and the patient with AIHA of mixed-type showed haemolytic activity and high numbers of PS exposing RBCs. Patients with decompensated AIHA appear to respond to treatment with erythropoietin, which is a known inhibitor of eryptosis.Eryptosis may frequently occur in AIHA related to IgM or IgA aab. Inhibition of eryptosis may represent a new therapeutic option in the treatment of AIHA. This article is protected by copyright. All rights reserved.
Pub.: 30 Sep '17, Pinned: 01 Oct '17
Abstract: Similar to nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which is characterized by cell shrinkage, cell membrane blebbing and cell membrane phospholipid scrambling. Eryptotic cells are removed and thus prevented from undergoing hemolysis. Eryptosis is stimulated by Ca(2+) following Ca(2+) entry through unspecific cation channels. Ca(2+) sensitivity is enhanced by ceramide, a product of acid sphingomyelinase. Eryptosis is triggered by hyperosmolarity, oxidative stress, energy depletion, hyperthermia and a wide variety of xenobiotics and endogenous substances. Eryptosis is inhibited by nitric oxide, catecholamines and a variety of further small molecules. Erythropoietin counteracts eryptosis in part by inhibiting the Ca(2+)-permeable cation channels but by the same token may foster formation of erythrocytes, which are particularly sensitive to eryptotic stimuli. Eryptosis is triggered in several clinical conditions such as iron deficiency, diabetes, renal insufficiency, myelodysplastic syndrome, phosphate depletion, sepsis, haemolytic uremic syndrome, mycoplasma infection, malaria, sickle-cell anemia, beta-thalassemia, glucose-6-phosphate dehydrogenase-(G6PD)-deficiency, hereditary spherocytosis, paroxysmal nocturnal hemoglobinuria, and Wilson's disease. Enhanced eryptosis is observed in mice with deficient annexin 7, cGMP-dependent protein kinase type I (cGKI), AMP-activated protein kinase AMPK, anion exchanger AE1, adenomatous polyposis coli APC and Klotho as well as in mouse models of sickle cell anemia and thalassemia. Eryptosis is decreased in mice with deficient phosphoinositide dependent kinase PDK1, platelet activating factor receptor, transient receptor potential channel TRPC6, janus kinase JAK3 or taurine transporter TAUT. If accelerated eryptosis is not compensated by enhanced erythropoiesis, clinically relevant anemia develops. Eryptotic erythrocytes may further bind to endothelial cells and thus impede microcirculation.
Pub.: 09 May '12, Pinned: 26 Sep '17
Abstract: The current paradigm for managing infectious diseases has targeted unique processes or enzymes within pathogens. A serious disadvantage of this pathogen-directed drug targeting strategy has been the development of microbial drug resistance and consequent resurgence of once-contained infectious diseases. A new drug discovery paradigm has therefore emerged focusing on identifying and targeting host factors essential for pathogen entry, survival, and replication. Innovative strategies combining genome-wide computational biology, genomics, proteomics, and traditional forward and reverse genetics have identified host-pathogen interactions and host functions critical for the establishment of infection. Chemogenomics and chemical genetics have allowed rapid identification of new and existing licensed drugs with antimicrobial activity. Although most host-directed drug targeting studies have focused on viral infections, they have provided a proof of concept for similar approaches to bacterial and parasite infections. Future therapies may combine conventional targeting of microbial virulence factors, together with host-directed drug therapy and augmentation of protective host factors, to efficiently eliminate the invading pathogen.
Pub.: 24 Jul '08, Pinned: 26 Sep '17
Abstract: Infectious diseases are a leading cause of global human mortality. The use of antimicrobials remains the most common strategy for treatment. However, the isolation of pathogens resistant to virtually all antimicrobials makes it urgent to develop effective therapeutics based on new targets. Here we review a new drug discovery paradigm focusing on identifying and targeting host factors important for infection as well as pathogen determinants involved in disease progression. We summarize innovative strategies which by combining bioinformatics with transcriptomics and chemical genetics have already identified host factors essential for pathogen entry, survival and replication. We describe how the discovery of RNA interference which allows loss-of-function studies has facilitated functional genomic studies in human cells. It is expected that these studies will identify targets to be used as host-directed drug therapy which, together with antimicrobials targeting microbial virulence factors, will efficiently eliminate the invading pathogen.
Pub.: 01 Apr '10, Pinned: 26 Sep '17