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151. Experimentally controlled downregulation of the histone chaperone FACT in Plasmodium berghei reveals that it is critical to male gamete fertility.

Author

Laurentino, Eliane C., Taylor, Sonya, Mair, Gunnar R., Lasonder, Edwin, Bartfai, Richard, Stunnenberg, Hendrik G., Kroeze, Hans, Ramesar, Jai, Franke-Fayard, Blandine, Khan, Shahid M., Janse, Chris J., and Waters, Andrew P.

Subjects
BIOLOGY experiments, HISTONES, MOLECULAR chaperones, SPERMATOZOA, CHROMATIN, PLASMODIUM, GENE expression, RNA polymerases, GERM cells
Abstract

Summary Human FACT (facilitates chromatin transcription) consists of the proteins SPT16 and SSRP1 and acts as a histone chaperone in the (dis)assembly of nucleosome (and thereby chromatin) structure during transcription and DNA replication. We identified a Plasmodium berghei protein, termed FACT-L, with homology to the SPT16 subunit of FACT. Epitope tagging of FACT-L showed nuclear localization with high expression in the nuclei of (activated) male gametocytes. The gene encoding FACT-L could not be deleted indicating an essential role during blood-stage development. Using a 'promoter-swap' approach whereby the fact-l promoter was replaced by an 'asexual blood stage-specific' promoter that is silent in gametocytes, transcription of fact-l in promoter-swap mutant gametocytes was downregulated compared with wild-type gametocytes. These mutant male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility was strongly reduced while female gamete fertility was unaffected; residual ookinetes generated oocysts that arrested early in development and failed to enter sporogony. Therefore FACT is critically involved in the formation of fertile male gametes and parasite transmission. 'Promoter swapping' is a powerful approach for the functional analysis of proteins in gametocytes (and beyond) that are essential during asexual blood-stage development. [ABSTRACT FROM AUTHOR]

Published
2011
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152. Investigating the evolution of apoptosis inmalaria parasites: the importance of ecology.

Author

Pollitt, Laura C., Colegrave, Nick, Khan, Shahid M., Sajid, Mohammed, and Reece, Sarah E.

Subjects
APOPTOSIS, CELL death, PROTOZOAN diseases, PLASMODIUM, PLASMODIIDAE, POPULATION biology, MALARIA, UNICELLULAR organisms, DEATH (Biology)
Abstract

Apoptosis is a precisely regulated process of cell death which occurs widely in multicellular organisms and is essential for normal development and immune defences. In recent years, interest has grown in the occurrence of apoptosis in unicellular organisms. In particular, as apoptosis has been reported in a wide range of species, including protozoan malaria parasites and trypanosomes, it may provide a novel target for intervention. However, it is important to understand when and why parasites employ an apoptosis strategy before the likely long- and shortterm success of such an intervention can be evaluated. The occurrence of apoptosis in unicellular parasites provides a challenge for evolutionary theory to explain as organisms are expected to have evolved to maximise their own proliferation, not death. One possible explanation is that protozoan parasites undergo apoptosis in order to gain a group benefit from controlling their density as this prevents premature vector mortality. However, experimental manipulations to examine the ultimate causes behind apoptosis in parasites are lacking. In this review, we focus on malaria parasites to outline how an evolutionary framework can help make predictions about the ecological circumstances under which apoptosis could evolve. We then highlight the ecological considerations that should be taken into account when designing evolutionary experiments involving markers of cell death, and we call for collaboration between researchers in different fields to identify and develop appropriate markers in reference to parasite ecology and to resolve debates on terminology. [ABSTRACT FROM AUTHOR]

Published
2010
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153. The Subcellular Location of Ovalbumin in Plasmodium bergheiBlood Stages Influences the Magnitude of T-Cell Responses

Author

Lin, Jing-Wen, Shaw, Tovah N., Annoura, Takeshi, Fougère, Aurélie, Bouchier, Pascale, Chevalley-Maurel, Séverine, Kroeze, Hans, Franke-Fayard, Blandine, Janse, Chris J., Couper, Kevin N., and Khan, Shahid M.

Abstract

ABSTRACTModel antigens are frequently introduced into pathogens to study determinants that influence T-cell responses to infections. To address whether an antigen's subcellular location influences the nature and magnitude of antigen-specific T-cell responses, we generated Plasmodium bergheiparasites expressing the model antigen ovalbumin (OVA) either in the parasite cytoplasm or on the parasitophorous vacuole membrane (PVM). For cytosolic expression, OVA alone or conjugated to mCherry was expressed from a strong constitutive promoter (OVAhsp70or OVA::mCherryhsp70); for PVM expression, OVA was fused to HEP17/EXP1 (OVA::Hep17hep17). Unexpectedly, OVA expression in OVAhsp70parasites was very low, but when OVA was fused to mCherry (OVA::mCherryhsp70), it was highly expressed. OVA expression in OVA::Hep17hep17parasites was strong but significantly less than that in OVA::mCherryhsp70parasites. These transgenic parasites were used to examine the effects of antigen subcellular location and expression level on the development of T-cell responses during blood-stage infections. While all OVA-expressing parasites induced activation and proliferation of OVA-specific CD8+T cells (OT-I) and CD4+T cells (OT-II), the level of activation varied: OVA::Hep17hep17parasites induced significantly stronger splenic and intracerebral OT-I and OT-II responses than those of OVA::mCherryhsp70parasites, but OVA::mCherryhsp70parasites promoted stronger OT-I and OT-II responses than those of OVAhsp70parasites. Despite lower OVA expression levels, OVA::Hep17hep17parasites induced stronger T-cell responses than those of OVA::mCherryhsp70parasites. These results indicate that unconjugated cytosolic OVA is not stably expressed in Plasmodiumparasites and, importantly, that its cellular location and expression level influence both the induction and magnitude of parasite-specific T-cell responses. These parasites represent useful tools for studying the development and function of antigen-specific T-cell responses during malaria infection.

Published
2014
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154. Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparumSporozoite Development in the Midgut of AnophelesMosquitoes

Author

van Schaijk, Ben C. L., Kumar, T. R. Santha, Vos, Martijn W., Richman, Adam, van Gemert, Geert-Jan, Li, Tao, Eappen, Abraham G., Williamson, Kim C., Morahan, Belinda J., Fishbaugher, Matt, Kennedy, Mark, Camargo, Nelly, Khan, Shahid M., Janse, Chris J., Sim, Kim Lee, Hoffman, Stephen L., Kappe, Stefan H. I., Sauerwein, Robert W., Fidock, David A., and Vaughan, Ashley M.

Abstract

ABSTRACTThe prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodiumparasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novovia the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum, the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparumby assessing the functional consequences of deleting fabIand fabB/F. Targeted deletion or disruption of these genes in P. falciparumdid not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparumFAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodiumparasites P. bergheiand P. yoelii, where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anophelesmosquito midgut.

Published
2014
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155. Reduced CD36-dependent tissue sequestration of Plasmodium-infected erythrocytes is detrimental to malaria parasite growth in vivo

Author

Fonager, Jannik, Pasini, Erica M., Braks, Joanna A.M., Klop, Onny, Ramesar, Jai, Remarque, Edmond J., Vroegrijk, Irene O.C.M., van Duinen, Sjoerd G., Thomas, Alan W., Khan, Shahid M., Mann, Matthias, Kocken, Clemens H.M., Janse, Chris J., and Franke-Fayard, Blandine M.D.

Abstract

Adherence of parasite-infected red blood cells (irbc) to the vascular endothelium of organs plays a key role in the pathogenesis of Plasmodium falciparum malaria. The prevailing hypothesis of why irbc adhere and sequester in tissues is that this acts as a mechanism of avoiding spleen-mediated clearance. Irbc of the rodent parasite Plasmodium berghei ANKA sequester in a fashion analogous to P. falciparum by adhering to the host receptor CD36. To experimentally determine the significance of sequestration for parasite growth, we generated a mutant P. berghei ANKA parasite with a reduced CD36-mediated adherence. Although the cognate parasite ligand binding to CD36 is unknown, we show that nonsequestering parasites have reduced growth and we provide evidence that in addition to avoiding spleen removal, other factors related to CD36-mediated sequestration are beneficial for parasite growth. These results reveal for the first time the importance of sequestration to a malaria infection, with implications for the development of strategies aimed at reducing pathology by inhibiting tissue sequestration.

Published
2012
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156. CCR4-Associated Factor 1 Coordinates the Expression of Plasmodium falciparumEgress and Invasion Proteins

Author

Balu, Bharath, Maher, Steven P., Pance, Alena, Chauhan, Chitra, Naumov, Anatoli V., Andrews, Robert M., Ellis, Peter D., Khan, Shahid M., Lin, Jing-wen, Janse, Chris J., Rayner, Julian C., and Adams, John H.

Abstract

ABSTRACTCoordinated regulation of gene expression is a hallmark of the Plasmodium falciparumasexual blood-stage development cycle. We report that carbon catabolite repressor protein 4 (CCR4)-associated factor 1 (CAF1) is critical in regulating more than 1,000 genes during malaria parasites' intraerythrocytic stages, especially egress and invasion proteins. CAF1 knockout results in mistimed expression, aberrant accumulation and localization of proteins involved in parasite egress, and invasion of new host cells, leading to premature release of predominantly half-finished merozoites, drastically reducing the intraerythrocytic growth rate of the parasite. This study demonstrates that CAF1 of the CCR4-Not complex is a significant gene regulatory mechanism needed for Plasmodiumdevelopment within the human host.

Published
2011
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157. Successful Transplantation of Kidneys from Deceased Donors with Acute Renal Failure Three-Year Results

Author

Kumar, Mysore S. Anil, Khan, Shahid M., Jaglan, Sandeep, Heifets, Michael, Moritz, Michael J., Saeed, Muhammad I., Fyfe, Billie, Sustento-Reodica, Nedjema, and Kumar, Aparna

Abstract

Kidneys from deceased donors with acute renal failure (ARF) are generally not accepted for transplantation because of the expected poor outcome. This prospective study examined the utilization of kidneys from donors with ARF for transplantation and the outcomes.

Published
2006
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158. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

159. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

160. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

162. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

163. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

164. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

165. Additional file 5 of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
3. Good health
Abstract

Additional file 5: List of primers used in this study. Left column: primer number. Middle column: Primer sequence. Right column: Description of primer and orientation (F: Forward, R: Reverse). (PDF 63 KB)

167. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

168. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

171. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

172. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

173. Additional file 5 of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
3. Good health
Abstract

Additional file 5: List of primers used in this study. Left column: primer number. Middle column: Primer sequence. Right column: Description of primer and orientation (F: Forward, R: Reverse). (PDF 63 KB)

175. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

177. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

178. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

179. Additional file 4 of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
3. Good health
Abstract

Additional file 4: Location of intron within the 5'ITR of piggyBac. Green letters: gfp CDS. Black underlined letters: piggyBac 5' ITR. Black underlined bold letters: Intron. (PDF 17 KB)

181. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

182. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

183. Additional file 4 of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
3. Good health
Abstract

Additional file 4: Location of intron within the 5'ITR of piggyBac. Green letters: gfp CDS. Black underlined letters: piggyBac 5' ITR. Black underlined bold letters: Intron. (PDF 17 KB)

185. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

186. Imaging of the spleen in malaria

Author

Ferrer, Mireia, Martin-Jaular, Lorena, De Niz Hidalgo, Mariana Isabel, Khan, Shahid M, Janse, Chris J, Calvo, Maria, Heussler, Volker, and Del Portillo, Hernando A

Subjects
570 Life sciences, biology, 3. Good health
Abstract

Splenomegaly, albeit variably, is a hallmark of malaria; yet, the role of the spleen in Plasmodium infections remains vastly unknown. The implementation of imaging to study the spleen is rapidly advancing our knowledge of this so-called "blackbox" of the abdominal cavity. Not only has ex vivo imaging revealed the complex functional compartmentalization of the organ and immune effector cells, but it has also allowed the observation of major structural remodeling during infections. In vivo imaging, on the other hand, has allowed quantitative measurements of the dynamic passage of the parasite at spatial and temporal resolution. Here, we review imaging techniques used for studying the malarious spleen, from optical microscopy to in vivo imaging, and discuss the bright perspectives of evolving technologies in our present understanding of the role of this organ in infections caused by Plasmodium.

187. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

188. A Plasmodium phospholipase is involved in disruption of the liver stage parasitophorous vacuole membrane

Author

Burda, Paul-Christian, Rölli, Matthias, Schaffner, Marco, Khan, Shahid M, Janse, Chris J, and Heussler, Volker

Subjects
parasitic diseases, 570 Life sciences, biology, 500 Science, 610 Medicine & health, 3. Good health
Abstract

The coordinated exit of intracellular pathogens from host cells is a process critical to the success and spread of an infection. While phospholipases have been shown to play important roles in bacteria host cell egress and virulence, their role in the release of intracellular eukaryotic parasites is largely unknown. We examined a malaria parasite protein with phospholipase activity and found it to be involved in hepatocyte egress. In hepatocytes, Plasmodium parasites are surrounded by a parasitophorous vacuole membrane (PVM), which must be disrupted before parasites are released into the blood. However, on a molecular basis, little is known about how the PVM is ruptured. We show that Plasmodium berghei phospholipase, PbPL, localizes to the PVM in infected hepatocytes. We provide evidence that parasites lacking PbPL undergo completely normal liver stage development until merozoites are produced but have a defect in egress from host hepatocytes. To investigate this further, we established a live-cell imaging-based assay, which enabled us to study the temporal dynamics of PVM rupture on a quantitative basis. Using this assay we could show that PbPL-deficient parasites exhibit impaired PVM rupture, resulting in delayed parasite egress. A wild-type phenotype could be re-established by gene complementation, demonstrating the specificity of the PbPL deletion phenotype. In conclusion, we have identified for the first time a Plasmodium phospholipase that is important for PVM rupture and in turn for parasite exit from the infected hepatocyte and therefore established a key role of a parasite phospholipase in egress.

189. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

190. Additional file 3 of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
3. Good health
Abstract

Additional file 3: Generation of parasites lacking expression of P41 (Δ p41 ) or Metacaspase2 (Δ metacaspase2 ). (A) Schematic representation of the construct used for disruption of p41 and metacaspase2. Correct integration of the construct results in disruption of the genes as shown (replacement locus) and was analysed by Southern analysis of PFG-separated chromosomes and diagnostic PCR (see B and C). See (Additional file 5 Table S3) for primer details. Black boxes: target regions; grey box: tgdhfr-ts selectable marker cassette. (B) PCR analysis of correct disruption of p41, Northern analysis of transcription in wild type (wt) and mutant (318cl1) and Southern analysis of PFG-separated chromosomes. PCRs were performed with primers that amplify the 5' (INT1 and L313) region of the disrupted locus (5' int) or the intact ORF (INT1, INT2), c: Control PCR amplifying the p28 gene. Chromosomes were hybridized with the 3'-dhfr probe recognizing the p41-construct integrated in chromosome 10 and the endogenous dhfr-ts gene on chromosome 7. TM4 is a probe recognizing rRNA and used as a loading control. (C) PCR analysis of correct disruption of the metacaspase2 and Southern analysis of PFG-separated chromosomes in wild type (wt) and mutant (796cl1) (right-hand panel). PCRs were performed with primers that amplify the 5' (INT1 and 313) region of the disrupted locus (5' int) or the intact ORF (INT1, INT2), c: Control PCR amplifying the p28 gene. Chromosomes were hybridized with the 3'-dhfr probe recognizing the metacaspase2 construct integrated in chromosome 10, the endogenous dhfr-ts gene on chromosome 7 and the integrated GFP-construct in chromosome 3 of the parent line 507cl1. (PDF 147 KB)

191. Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
animal structures, fungi, parasitic diseases, 3. Good health
Abstract

Additional file of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

192. Additional file 3 of Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Author

Fonager, Jannik, Blandine MD Franke-Fayard, Adams, John H, Ramesar, Jai, Onny Klop, Khan, Shahid M, Janse, Chris J, and Waters, Andrew P

Subjects
3. Good health
Abstract

Additional file 3: Generation of parasites lacking expression of P41 (Δ p41 ) or Metacaspase2 (Δ metacaspase2 ). (A) Schematic representation of the construct used for disruption of p41 and metacaspase2. Correct integration of the construct results in disruption of the genes as shown (replacement locus) and was analysed by Southern analysis of PFG-separated chromosomes and diagnostic PCR (see B and C). See (Additional file 5 Table S3) for primer details. Black boxes: target regions; grey box: tgdhfr-ts selectable marker cassette. (B) PCR analysis of correct disruption of p41, Northern analysis of transcription in wild type (wt) and mutant (318cl1) and Southern analysis of PFG-separated chromosomes. PCRs were performed with primers that amplify the 5' (INT1 and L313) region of the disrupted locus (5' int) or the intact ORF (INT1, INT2), c: Control PCR amplifying the p28 gene. Chromosomes were hybridized with the 3'-dhfr probe recognizing the p41-construct integrated in chromosome 10 and the endogenous dhfr-ts gene on chromosome 7. TM4 is a probe recognizing rRNA and used as a loading control. (C) PCR analysis of correct disruption of the metacaspase2 and Southern analysis of PFG-separated chromosomes in wild type (wt) and mutant (796cl1) (right-hand panel). PCRs were performed with primers that amplify the 5' (INT1 and 313) region of the disrupted locus (5' int) or the intact ORF (INT1, INT2), c: Control PCR amplifying the p28 gene. Chromosomes were hybridized with the 3'-dhfr probe recognizing the metacaspase2 construct integrated in chromosome 10, the endogenous dhfr-ts gene on chromosome 7 and the integrated GFP-construct in chromosome 3 of the parent line 507cl1. (PDF 147 KB)

194. Pre-clinical evaluation of a P. berghei-based whole-sporozoite malaria vaccine candidate.

Author

Mendes, António M., Reuling, Isaie J., Andrade, Carolina M., Otto, Thomas D., Machado, Marta, Teixeira, Filipa, Pissarra, Joana, Gonçalves-Rosa, Nataniel, Bonaparte, Dolores, Sinfrónio, João, Sanders, Mandy, Janse, Chris J., Khan, Shahid M., Newbold, Chris I., Berriman, Matthew, Lee, Cynthia K., Wu, Yimin, Ockenhouse, Christian F., Sauerwein, Robert W., and Prudêncio, Miguel

Subjects
PLASMODIUM berghei, SPOROZOITES, MALARIA vaccines, PLASMODIUM falciparum, CIRCUMSPOROZOITE protein
Abstract

Whole-sporozoite vaccination/immunization induces high levels of protective immunity in both rodent models of malaria and in humans. Recently, we generated a transgenic line of the rodent malaria parasite P. berghei (Pb) that expresses the P. falciparum (Pf) circumsporozoite protein (PfCS), and showed that this parasite line (PbVac) was capable of (1) infecting and developing in human hepatocytes but not in human erythrocytes, and (2) inducing neutralizing antibodies against the human Pf parasite. Here, we analyzed PbVac in detail and developed tools necessary for its use in clinical studies. A microbiological contaminant-free Master Cell Bank of PbVac parasites was generated through a process of cyclic propagation and clonal expansion in mice and mosquitoes and was genetically characterized. A highly sensitive qRT-PCR-based method was established that enables PbVac parasite detection and quantification at low parasite densities in vivo. This method was employed in a biodistribution study in a rabbit model, revealing that the parasite is only present at the site of administration and in the liver up to 48 h post infection and is no longer detectable at any site 10 days after administration. An extensive toxicology investigation carried out in rabbits further showed the absence of PbVac-related toxicity. In vivo drug sensitivity assays employing rodent models of infection showed that both the liver and the blood stage forms of PbVac were completely eliminated by Malarone® treatment. Collectively, our pre-clinical safety assessment demonstrates that PbVac possesses all characteristics necessary to advance into clinical evaluation. Malaria: Pre-clinical characterization of transgenic malaria vaccine PbVac is a transgenic malaria parasite expressing circumsporozoite antigen from the human parasite Plasmodium falciparum. PbVac elicits neutralizing P. falciparum antibodies and can infect human hepatocytes but not erythrocytes, suggesting that humans would be non-permissive. Miguel Prudêncio and colleagues at the Institute of Molecular Medicine in Lisbon perform a detailed in vivo analysis and toxicology of PbVac. Extensive biodistribution analysis using a highly sensitive qPCR in non-permissive rabbit hosts shows PbVac are present at the initial bite site early on with later appearance in the liver, but by day 10 is undetectable. Importantly no PbVac could be detected in the blood at any time-point. PbVac was well tolerated with no apparent pathological signatures. In permissive mouse hosts PbVac could be effectively eliminated from both the blood and liver and could thereby act as a potential clinical 'safety net' in the event of an erythrocytic stage or persistence in the liver. [ABSTRACT FROM AUTHOR]

Published
2018
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195. The Plasmodium falciparum male gametocyte protein P230p, a paralog of P230, is vital for ookinete formation and mosquito transmission.

Author

Marin-Mogollon, Catherin, van de Vegte-Bolmer, Marga, van Gemert, Geert-Jan, van Pul, Fiona J. A., Ramesar, Jai, Othman, Ahmad Syibli, Kroeze, Hans, Miao, Jun, Cui, Liwang, Williamson, Kim C., Sauerwein, Robert W., Janse, Chris J., and Khan, Shahid M.

Abstract

Two members of 6-cysteine (6-cys) protein family, P48/45 and P230, are important for gamete fertility in rodent and human malaria parasites and are leading transmission blocking vaccine antigens. Rodent and human parasites encode a paralog of P230, called P230p. While P230 is expressed in male and female parasites, P230p is expressed only in male gametocytes and gametes. In rodent malaria parasites this protein is dispensable throughout the complete life-cycle; however, its function in P. falciparum is unknown. Using CRISPR/Cas9 methodology we disrupted the gene encoding Pfp230p resulting in P. falciparum mutants (PfΔp230p) lacking P230p expression. The PfΔp230p mutants produced normal numbers of male and female gametocytes, which retained expression of P48/45 and P230. Upon activation male PfΔp230p gametocytes undergo exflagellation and form male gametes. However, male gametes are unable to attach to red blood cells resulting in the absence of characteristic exflagellation centres in vitro. In the absence of P230p, zygote formation as well as oocyst and sporozoite development were strongly reduced (>98%) in mosquitoes. These observations demonstrate that P230p, like P230 and P48/45, has a vital role in P. falciparum male fertility and zygote formation and warrants further investigation as a potential transmission blocking vaccine candidate. [ABSTRACT FROM AUTHOR]

Published
2018
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196. Chimeric Plasmodium falciparum parasites expressing Plasmodium vivax circumsporozoite protein fail to produce salivary gland sporozoites.

Author

Marin-Mogollon, Catherin, van Pul, Fiona J. A., Miyazaki, Shinya, Imai, Takashi, Ramesar, Jai, Salman, Ahmed M., Winkel, Beatrice M. F., Othman, Ahmad Syibli, Kroeze, Hans, Chevalley-Maurel, Severine, Reyes-Sandoval, Arturo, Roestenberg, Meta, Franke-Fayard, Blandine, Janse, Chris J., and Khan, Shahid M.

Subjects
PLASMODIUM genetics, CIRCUMSPOROZOITE protein, PLASMODIUM, PLASMODIUM falciparum, PLASMODIUM vivax, GENOME editing, ALLELES, SPOROZOITES
Abstract

Background: Rodent malaria parasites where the gene encoding circumsporozoite protein (CSP) has been replaced with csp genes from the human malaria parasites, Plasmodium falciparum or Plasmodium vivax, are used as pre-clinical tools to evaluate CSP vaccines in vivo. These chimeric rodent parasites produce sporozoites in Anopheles stephensi mosquitoes that are capable of infecting rodent and human hepatocytes. The availability of chimeric P. falciparum parasites where the pfcsp gene has been replaced by the pvcsp would open up possibilities to test P. vivax CSP vaccines in small scale clinical trials using controlled human malaria infection studies. Methods: Using CRISPR/Cas9 gene editing two chimeric P. falciparum parasites, were generated, where the pfcsp gene has been replaced by either one of the two major pvcsp alleles, VK210 or VK247. In addition, a P. falciparum parasite line that lacks CSP expression was also generated. These parasite lines have been analysed for sporozoite production in An. stephensi mosquitoes. Results: The two chimeric Pf-PvCSP lines exhibit normal asexual and sexual blood stage development in vitro and produce sporozoite-containing oocysts in An. stephensi mosquitoes. Expression of the corresponding PvCSP was confirmed in oocyst-derived Pf-PvCSP sporozoites. However, most oocysts degenerate before sporozoite formation and sporozoites were not found in either the mosquito haemocoel or salivary glands. Unlike the chimeric Pf-PvCSP parasites, oocysts of P. falciparum parasites lacking CSP expression do not produce sporozoites. Conclusions: Chimeric P. falciparum parasites expressing P. vivax circumsporozoite protein fail to produce salivary gland sporozoites. Combined, these studies show that while PvCSP can partially complement the function of PfCSP, species-specific features of CSP govern full sporozoite maturation and development in the two human malaria parasites. [ABSTRACT FROM AUTHOR]

Published
2018
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197. Preclinical Development and Assessment of Viral Vectors Expressing a Fusion Antigen of Plasmodium falciparumLSA1 and LSAP2 for Efficacy against Liver-Stage Malaria

Author

Halbroth, Benedict R., Sebastian, Sarah, Salman, Ahmed M., Ulaszewska, Marta, Gola, Anita, Longley, Rhea J., Janse, Chris J., Khan, Shahid M., Hill, Adrian V. S., and Spencer, Alexandra J.

Abstract

Despite promising progress in malaria vaccine development in recent years, an efficacious subunit vaccine against Plasmodium falciparumremains to be licensed and deployed. Cell-mediated protection from liver-stage malaria relies on a sufficient number of antigen-specific T cells reaching the liver during the time that parasites are present. A single vaccine expressing two antigens could potentially increase both the size and breadth of the antigen-specific response while halving vaccine production costs.

Published
2019
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198. Self-organization: Two's company, three's a crowd.

Author

Khan, Shahid M. and Molloy, Justin E.

Subjects
*SELF-organizing systems, *BIOPOLYMERS, *BIOMOLECULES, *ACTIN research, *DISTRIBUTION (Probability theory)
Abstract

The article discusses research on the physical rules governing self-organization of biopolymers in cells observed using real-time tracking of self-propelled biomolecules. The study recorded the movement of individual actin filaments during cell division and analyzed the probability distribution for the incident angle made between colliding filaments, deviation between incoming and outgoing angles, probability of binary filament collisions, and ordered phase transition at filament densities.

Published
2015
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199. Prime and target immunization protects against liver-stage malaria in mice

Author

Gola, Anita, Silman, Daniel, Walters, Adam A., Sridhar, Saranya, Uderhardt, Stefan, Salman, Ahmed M., Halbroth, Benedict R., Bellamy, Duncan, Bowyer, Georgina, Powlson, Jonathan, Baker, Megan, Venkatraman, Navin, Poulton, Ian, Berrie, Eleanor, Roberts, Rachel, Lawrie, Alison M., Angus, Brian, Khan, Shahid M., Janse, Chris J., Ewer, Katie J., Germain, Ronald N., Spencer, Alexandra J., and Hill, Adrian V. S.

Abstract

A prime and target vaccine for liver-stage malaria results in sterile protection in mouse disease models and appears safe for human use.

Published
2018
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200. Tailoring a Plasmodium vivaxVaccine To Enhance Efficacy through a Combination of a CSP Virus-Like Particle and TRAP Viral Vectors

Author

Atcheson, Erwan, Bauza, Karolis, Salman, Ahmed M., Alves, Eduardo, Blight, Joshua, Viveros-Sandoval, Martha Eva, Janse, Chris J., Khan, Shahid M., Hill, Adrian V. S., and Reyes-Sandoval, Arturo

Abstract

Vivax malaria remains one of the most serious and neglected tropical diseases, with 132 to 391 million clinical cases per year and 2.5 billion people at risk of infection. A vaccine against Plasmodium vivaxcould have more impact than any other intervention, and the use of a vaccine targeting multiple antigens may result in higher efficacy against sporozoite infection than targeting a single antigen.

Published
2018
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