scholarly journals A Second Mechanism Employed by Artemisinins to Suppress Plasmodium Falciparum Hinges on Inhibition of Hematin Crystallization

2020 ◽  
pp. jbc.RA120.016115
Author(s):  
Wenchuan Ma ◽  
Victoria A. Balta ◽  
Rachel West ◽  
Katy N. Newlin ◽  
Ognjen Š. Miljanić ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Parent Drug ◽  
Parasite Life Cycle ◽  
Drug Dosing ◽  
Low Concentrations ◽  
Carbon Centered Radical ◽  
Dosing Regimens ◽  
Drug Activation

Malaria is a pervasive disease that affects millions of lives each year in equatorial regions of the world. During the erythrocytic phase of the parasite life cycle, Plasmodium falciparum invade red blood cells, where they catabolize hemoglobin and sequester the released toxic heme as innocuous hemozoin crystals. Artemisinin-class drugs are activated in vivo by newly-released heme, which creates a carbon-centered radical that markedly reduces parasite density. Radical damage to parasite lipids and proteins is perceived to be artemisinins’ dominant mechanism of action. By contrast, quinoline-class antimalarials inhibit the formation of hemozoin and in this way suppress heme detoxification. Here, we combine malaria parasite assays and scanning probe microscopy of growing beta-hematin crystals to elucidate an unexpected mechanism employed by two widely administered antimalarials, artemisinin and artesunate, to subdue the erythrocytic phase of the parasite life cycle. We demonstrate that heme-drug adducts, produced after the radical activation of artemisinins and largely believed to be benign bystanders, potently kills P. falciparum at low concentrations. We show that these adducts inhibit b-hematin crystallization and heme detoxification, a pathway which complements the deleterious effect of radicals generated via parent drug activation. Our findings reveal an irreversible mechanism of heme-artemisinin adduct inhibition of heme crystallization, unique among antimalarials and common crystal growth inhibitors, that opens new avenues for evaluating drug dosing regimens and understanding growing resistance of P. falciparum to artemisinin.

A transgenic Plasmodium falciparum NF54 strain that expresses GFP–luciferase throughout the parasite life cycle

2012 ◽  
Vol 186 (2) ◽  
pp. 143-147 ◽  
Author(s):  
Ashley M. Vaughan ◽  
Sebastian A. Mikolajczak ◽  
Nelly Camargo ◽  
Viswanathan Lakshmanan ◽  
Mark Kennedy ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Parasite Life Cycle

scholarly journals RNase III Domain of KREPB9 and KREPB10 Association with Editosomes in Trypanosoma brucei

mSphere ◽  
2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Jason Carnes ◽  
Suzanne M. McDermott ◽  
Kenneth Stuart
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Tsetse Fly ◽  
Accessory Proteins ◽  
Rnase Iii ◽  
Parasite Life Cycle ◽  
Null Cell ◽  
Content Type

ABSTRACT Editosomes are the multiprotein complexes that catalyze the insertion and deletion of uridines to create translatable mRNAs in the mitochondria of kinetoplastids. Recognition and cleavage of a broad diversity of RNA substrates in vivo require three functionally distinct RNase III-type endonucleases, as well as five additional editosome proteins that contain noncatalytic RNase III domains. RNase III domains have recently been identified in the editosome accessory proteins KREPB9 and KREPB10, suggesting a role related to editing endonuclease function. In this report, we definitively show that KREPB9 and KREPB10 are not essential in either bloodstream-form parasites (BF) or procyclic-form parasites (PF) by creating null or conditional null cell lines. While preedited and edited transcripts are largely unaffected by the loss of KREPB9 in both PF and BF, loss of KREPB10 produces distinct responses in BF and PF. BF cells lacking KREPB10 also lack edited CYb, while PF cells have increased edited A6, RPS12, ND3, and COII after loss of KREPB10. We also demonstrate that mutation of the RNase III domain of either KREPB9 or KREPB10 results in decreased association with ~20S editosomes. Editosome interactions with KREPB9 and KREPB10 are therefore mediated by the noncatalytic RNase III domain, consistent with a role in endonuclease specialization in Trypanosoma brucei. IMPORTANCE Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness. U insertion/deletion RNA editing in T. brucei generates mature mitochondrial mRNAs. Editing is essential for survival in mammalian hosts and tsetse fly vectors and is differentially regulated during the parasite life cycle. Three multiprotein “editosomes,” typified by exclusive RNase III endonucleases that act at distinct sites, catalyze editing. Here, we show that editosome accessory proteins KREPB9 and KREPB10 are not essential for mammalian blood- or insect-form parasite survival but have specific and differential effects on edited RNA abundance in different stages. We also characterize KREPB9 and KREPB10 noncatalytic RNase III domains and show they are essential for editosome association, potentially via dimerization with RNase III domains in other editosome proteins. This work enhances the understanding of distinct editosome and accessory protein functions, and thus differential editing, during the parasite life cycle and highlights the importance of RNase III domain interactions to editosome architecture.

scholarly journals Nanobody generation and structural characterization of Plasmodium falciparum 6-cysteine protein Pf12p

2021 ◽  
Vol 478 (3) ◽  
pp. 579-595
Author(s):  
Melanie H. Dietrich ◽  
Li-Jin Chan ◽  
Amy Adair ◽  
Sravya Keremane ◽  
Phillip Pymm ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Vaccine Development ◽  
Structural Information ◽  
Family Members ◽  
Structural Features ◽  
Protein Family ◽  
Parasite Life Cycle ◽  
D2 Domain

Surface-associated proteins play critical roles in the Plasmodium parasite life cycle and are major targets for vaccine development. The 6-cysteine (6-cys) protein family is expressed in a stage-specific manner throughout Plasmodium falciparum life cycle and characterized by the presence of 6-cys domains, which are β-sandwich domains with conserved sets of disulfide bonds. Although several 6-cys family members have been implicated to play a role in sexual stages, mosquito transmission, evasion of the host immune response and host cell invasion, the precise function of many family members is still unknown and structural information is only available for four 6-cys proteins. Here, we present to the best of our knowledge, the first crystal structure of the 6-cys protein Pf12p determined at 2.8 Å resolution. The monomeric molecule folds into two domains, D1 and D2, both of which adopt the canonical 6-cys domain fold. Although the structural fold is similar to that of Pf12, its paralog in P. falciparum, we show that Pf12p does not complex with Pf41, which is a known interaction partner of Pf12. We generated 10 distinct Pf12p-specific nanobodies which map into two separate epitope groups; one group which binds within the D2 domain, while several members of the second group bind at the interface of the D1 and D2 domain of Pf12p. Characterization of the structural features of the 6-cys family and their associated nanobodies provide a framework for generating new tools to study the diverse functions of the 6-cys protein family in the Plasmodium life cycle.

scholarly journals Exploring Drug Dosing Regimens In Vitro Using Real-Time 3D Spheroid Tumor Growth Assays

2017 ◽  
Vol 22 (5) ◽  
pp. 537-546 ◽  
Author(s):  
Madhu Lal-Nag ◽  
Lauren McGee ◽  
Steven A. Titus ◽  
Kyle Brimacombe ◽  
Sam Michael ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Drug Discovery ◽  
Tumor Growth ◽  
Real Time ◽  
Chemotherapeutic Agents ◽  
Cancer Drug ◽  
Drug Dosing ◽  
Dosing Regimens

Two-dimensional monolayer cell proliferation assays for cancer drug discovery have made the implementation of large-scale screens feasible but only seem to reflect a simplified view that oncogenes or tumor suppressor genes are the genetic drivers of cancer cell proliferation. However, there is now increased evidence that the cellular and physiological context in which these oncogenic events occur play a key role in how they drive tumor growth in vivo and, therefore, in how tumors respond to drug treatments. In vitro 3D spheroid tumor models are being developed to better mimic the physiology of tumors in vivo, in an attempt to improve the predictability and efficiency of drug discovery for the treatment of cancer. Here we describe the establishment of a real-time 3D spheroid growth, 384-well screening assay. The cells used in this study constitutively expressed green fluorescent protein (GFP), which enabled the real-time monitoring of spheroid formation and the effect of chemotherapeutic agents on spheroid size at different time points of sphere growth and drug treatment. This real-time 3D spheroid assay platform represents a first step toward the replication in vitro of drug dosing regimens being investigated in vivo. We hope that further development of this assay platform will allow the investigation of drug dosing regimens, efficacy, and resistance before preclinical and clinical studies.

scholarly journals Growth Rate of Plasmodium falciparum: Analysis of Parasite Growth Data from Malaria Volunteer Infection Studies

2019 ◽  
Author(s):  
Leesa F Wockner ◽  
Isabell Hoffmann ◽  
Lachlan Webb ◽  
Benjamin Mordmüller ◽  
Sean C Murphy ◽  
...  
Keyword(s):  
Growth Rate ◽  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Sine Wave ◽  
Parasite Growth ◽  
Cycle Duration ◽  
Multiplication Rate ◽  
Growth Data ◽  
Parasite Life Cycle ◽  
Log Linear

Abstract Background Growth rate of malaria parasites in the blood of infected subjects is an important measure of efficacy of drugs and vaccines. Methods We used log-linear and sine-wave models to estimate the parasite growth rate of the 3D7 strain of Plasmodium falciparum using data from 177 subjects from 14 induced blood stage malaria (IBSM) studies conducted at QIMR Berghofer. We estimated parasite multiplication rate per 48 hour (PMR48), PMR per life-cycle (PMRLC), and parasite life-cycle duration. We compared these parameters to those from studies conducted elsewhere with infections induced by IBSM (n=66), sporozoites via mosquito bite (n=336) or injection (n=51). Results The parasite growth rate of 3D7 in QIMR Berghofer studies was 0.75/day (95% CI: 0.73–0.77/day), PMR48 was 31.9 (95% CI: 28.7–35.4), PMRLC was 16.4 (95% CI: 15.1–17.8) and parasite life-cycle was 38.8 hour (95% CI: 38.3–39.2 hour). These parameters were similar to estimates from IBSM studies elsewhere (0.71/day, 95% CI: 0.67–0.75/day; PMR48 26.6, 95% CI: 22.2–31.8), but significantly higher (P < 0.001) than in sporozoite studies (0.47/day, 95% CI: 0.43–0.50/day; PMR48 8.6, 95% CI: 7.3–10.1). Conclusion Parasite growth rates were similar across different IBSM studies and higher than infections induced by sporozoite.

scholarly journals Nanobody generation and structural characterization of Plasmodium falciparum 6-cysteine protein Pf12p

2020 ◽  
Author(s):  
Melanie H Dietrich ◽  
Li-Jin Chan ◽  
Amy Adair ◽  
Sravya Keremane ◽  
Phillip Pymm ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Vaccine Development ◽  
Structural Information ◽  
Family Members ◽  
Structural Features ◽  
Protein Family ◽  
Parasite Life Cycle ◽  
D2 Domain

Surface-associated proteins play critical roles in the Plasmodium parasite life cycle and are major targets for vaccine development. The 6-cysteine (6-cys) protein family is expressed in a stage-specific manner throughout Plasmodium falciparum life cycle and characterized by the presence of 6-cys domains, which are β-sandwich domains with conserved sets of disulfide bonds. Although several 6-cys family members have been implicated to play a role in sexual stages, mosquito transmission, evasion of the host immune response and host cell invasion, the precise function of many family members is still unknown and structural information is only available for four 6-cys proteins. Here, we present to the best of our knowledge, the first crystal structure of the 6-cys protein Pf12p determined at 2.8 Å resolution. The monomeric molecule folds into two domains, D1 and D2, both of which adopt the canonical 6-cys domain fold. Although the structural fold is similar to that of Pf12, its paralog in P. falciparum, we show that Pf12p does not complex with Pf41, which is a known interaction partner of Pf12. We generated ten distinct Pf12p-specific nanobodies which map into two separate epitope groups; one group which binds within the D2 domain, while several members of the second group bind at the interface of the D1 and D2 domain of Pf12p. Characterization of the structural features of the 6-cys family and their associated nanobodies provide a framework for generating new tools to study the diverse functions of the 6-cys protein family in the Plasmodium life cycle.

scholarly journals Impact of Antimicrobial Dosing Regimen on Evolution of Drug Resistance In Vivo: Fluconazole and Candida albicans

2006 ◽  
Vol 50 (7) ◽  
pp. 2374-2383 ◽  
Author(s):  
D. Andes ◽  
A. Forrest ◽  
A. Lepak ◽  
J. Nett ◽  
K. Marchillo ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Resistant Cell ◽  
In Vivo Model ◽  
Dosing Regimen ◽  
Resistance Development ◽  
Drug Dosing ◽  
Dosing Regimens ◽  
Resistant Strains ◽  
Selection Of

ABSTRACT Numerous factors have been theorized to affect the development of antimicrobial resistance, including those specific to the host, the organism, the environment, the drug, and the drug prescriber. One variable under the control of the prescriber is the drug dosing regimen. Dosing regimens can vary in dose level, dosing interval, and treatment duration. The current studies examined the relationships between antimicrobial dosing regimens and resistance development by use of an in vivo model. A murine model of systemic Candida albicans infection was used to examine resistance emergence during exposure to the triazole antifungal fluconazole. Data from this experimental model demonstrated that the more frequently administered dosing prevented selection of the isogenic resistant cell populations. Conversely, dosing regimens producing prolonged sub-MIC effects appeared to contribute to the outgrowth of isogenic resistant strains. The association between dosing and resistance emergence observed in the current investigation is disparate from that described for antimicrobial compounds with cidal killing characteristics. The inhibitory or static antimicrobial activity of the triazole compounds may explain these differences.

Origin, kinetics of circulation and fate in vivo of the major excretory–secretory product of Acanthocheilonema viteae

Parasitology ◽  
1989 ◽  
Vol 99 (2) ◽  
pp. 229-239 ◽  
Author(s):  
W. Harnett ◽  
M. J. Worms ◽  
A. Kapil ◽  
M. Grainger ◽  
R. M. E. Parkhouse
Keyword(s):  
Molecular Weight ◽  
Life Cycle ◽  
Half Life ◽  
Secretory Product ◽  
Parasite Life Cycle ◽  
Acanthocheilonema Viteae ◽  
Sds Page ◽  
Kinetics Of ◽  
Immunohistological Analysis

SummaryThe excretions-secretions (E-S) of Acanthocheilonema viteae consist mainly of one product, molecular weight 62 kDa. This molecule is synthesized during the vertebrate phase of the parasite life-cycle and is first detectable in the E-S of L4 parasites. It is cross-reactive with E-S of human filarial parasites as a consequence of possessing a phosphorylcholine (PC) moiety. The 62 kDa molecule has been employed as a model for the study of the origin and fate of filarial E-S. Immunohistological analysis has shown the molecule to be located predominantly in the parasite gut. Transplantation of adult female [S] methionine pulsed worms into uninfected jirds resulted in the radio-labelled secreted 62 kDa antigen being detected in the bloodstream within 4 h by SDS–PAGE/immunoprecipitation analysis. The systemic half-life of the molecule as estimated by clearance of injected, purified I-labelled material was measured in naive and infected jird hosts.

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

1990 ◽  
Vol 48 (3) ◽  
pp. 914-915
Author(s):  
D.J.P. Ferguson ◽  
A.R. Berendt ◽  
J. Tansey ◽  
K. Marsh ◽  
C.I. Newbold
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Umbilical Vein ◽  
Cell Types ◽  
Amelanotic Melanoma ◽  
Cytoplasmic Process ◽  
Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

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