Probing Plasmodium falciparum sexual commitment at the single-cell level

Background: Malaria parasites go through major transitions during their complex life cycle, yet the underlying differentiation pathways remain obscure. Here we apply single cell transcriptomics to unravel the program inducing sexual differentiation in Plasmodium falciparum. Parasites have to make this essential life-cycle decision in preparation for human-to-mosquito transmission. Methods: By combining transcriptional profiling with quantitative imaging and genetics, we defined a transcriptional signature in sexually committed cells. Results: We found this transcriptional signature to be distinct from general changes in parasite metabolism that can be observed in response to commitment-inducing conditions. Conclusions: This proof-of-concept study provides a template to capture transcriptional diversity in parasite populations containing complex mixtures of different life-cycle stages and developmental programs, with important implications for our understanding of parasite biology and the ongoing malaria elimination campaign.

Download Full-text

Probing Plasmodium falciparum sexual commitment at the single-cell level

Wellcome Open Research ◽

10.12688/wellcomeopenres.14645.3 ◽

2018 ◽

Vol 3 ◽

pp. 70 ◽

Cited By ~ 1

Author(s):

Nicolas M.B. Brancucci ◽

Mariana De Niz ◽

Timothy J. Straub ◽

Deepali Ravel ◽

Lauriane Sollelis ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Life Cycle ◽

Single Cell ◽

Transcriptional Profiling ◽

Quantitative Imaging ◽

Complex Life Cycle ◽

Major Transitions ◽

Transcriptional Signature ◽

Life Cycle Stages ◽

Parasite Populations

Background: Malaria parasites go through major transitions during their complex life cycle, yet the underlying differentiation pathways remain obscure. Here we apply single cell transcriptomics to unravel the program inducing sexual differentiation in Plasmodium falciparum. Parasites have to make this essential life-cycle decision in preparation for human-to-mosquito transmission. Methods: By combining transcriptional profiling with quantitative imaging and genetics, we defined a transcriptional signature in sexually committed cells. Results: We found this transcriptional signature to be distinct from general changes in parasite metabolism that can be observed in response to commitment-inducing conditions. Conclusions: This proof-of-concept study provides a template to capture transcriptional diversity in parasite populations containing complex mixtures of different life-cycle stages and developmental programs, with important implications for our understanding of parasite biology and the ongoing malaria elimination campaign.

Download Full-text

Probing Plasmodium falciparum sexual commitment at the single-cell level

Wellcome Open Research ◽

10.12688/wellcomeopenres.14645.1 ◽

2018 ◽

Vol 3 ◽

pp. 70 ◽

Cited By ~ 1

Author(s):

Nicolas M.B. Brancucci ◽

Mariana De Niz ◽

Timothy J. Straub ◽

Deepali Ravel ◽

Lauriane Sollelis ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Life Cycle ◽

Single Cell ◽

Transcriptional Profiling ◽

Quantitative Imaging ◽

Complex Life Cycle ◽

Major Transitions ◽

Transcriptional Signature ◽

Life Cycle Stages ◽

Parasite Populations

Background: Malaria parasites go through major transitions during their complex life cycle, yet the underlying differentiation pathways remain obscure. Here we apply single cell transcriptomics to unravel the program inducing sexual differentiation in Plasmodium falciparum. Parasites have to make this essential life-cycle decision in preparation for human-to-mosquito transmission. Methods: By combining transcriptional profiling with quantitative imaging and genetics, we defined a transcriptional signature in sexually committed cells. Results: We found this transcriptional signature to be distinct from general changes in parasite metabolism that can be observed in response to commitment-inducing conditions. Conclusions: This proof-of-concept study provides a template to capture transcriptional diversity in parasite populations containing complex mixtures of different life-cycle stages and developmental programs, with important implications for our understanding of parasite biology and the ongoing malaria elimination campaign.

Download Full-text

A single-cell atlas of Plasmodium falciparum transmission through the mosquito

10.1101/2020.10.11.333179 ◽

2020 ◽

Author(s):

Eliana Real ◽

Virginia M. Howick ◽

Farah Dahalan ◽

Kathrin Witmer ◽

Juliana Cudini ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Life Cycle ◽

Single Cell ◽

Developmental Trajectories ◽

Complex Life Cycle ◽

Transmission Cycle ◽

Host Tropism ◽

Specific Host ◽

Gene Usage ◽

Species Specific

AbstractMalaria parasites have a complex life cycle featuring diverse developmental strategies, each uniquely adapted to navigate specific host environments. Here we use single-cell transcriptomics to illuminate gene usage across the transmission cycle of the most virulent agent of human malaria – Plasmodium falciparum. We reveal developmental trajectories associated with the colonisation of the mosquito midgut and salivary glands and elucidate the transcriptional signatures of each transmissible stage. Additionally, we identify both conserved and nonconserved gene usage between human and rodent parasites, which point to both essential mechanisms in malaria transmission and species-specific adaptations potentially linked to host tropism. Together, the data presented here, which are made freely available via an interactive website, establish the most complete atlas of the P. falciparum transcriptional journey to date.One sentence summarySingle-cell transcriptomics of P. falciparum transmission stages highlights developmental trajectories and gene usage.

Download Full-text

Autonomy and integration in complex parasite life cycles

Parasitology ◽

10.1017/s0031182016001311 ◽

2016 ◽

Vol 143 (14) ◽

pp. 1824-1846 ◽

Cited By ~ 10

Author(s):

DANIEL P. BENESH

Keyword(s):

Life Cycle ◽

Holistic Approach ◽

Life Cycles ◽

Complex Life Cycle ◽

Functional Specialization ◽

Life Stages ◽

Free Living ◽

New Host ◽

Complex Life Cycles ◽

Life Cycle Stages

SUMMARYComplex life cycles are common in free-living and parasitic organisms alike. The adaptive decoupling hypothesis postulates that separate life cycle stages have a degree of developmental and genetic autonomy, allowing them to be independently optimized for dissimilar, competing tasks. That is, complex life cycles evolved to facilitate functional specialization. Here, I review the connections between the different stages in parasite life cycles. I first examine evolutionary connections between life stages, such as the genetic coupling of parasite performance in consecutive hosts, the interspecific correlations between traits expressed in different hosts, and the developmental and functional obstacles to stage loss. Then, I evaluate how environmental factors link life stages through carryover effects, where stressful larval conditions impact parasites even after transmission to a new host. There is evidence for both autonomy and integration across stages, so the relevant question becomes how integrated are parasite life cycles and through what mechanisms? By highlighting how genetics, development, selection and the environment can lead to interdependencies among successive life stages, I wish to promote a holistic approach to studying complex life cycle parasites and emphasize that what happens in one stage is potentially highly relevant for later stages.

Download Full-text

A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the Plasmodium falciparum Life Cycle

mBio ◽

10.1128/mbio.01170-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 10

Author(s):

Marta Tibúrcio ◽

Annie S. P. Yang ◽

Kazuhide Yahata ◽

Pablo Suárez-Cortés ◽

Hugo Belda ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Life Cycle ◽

Genetic Modification ◽

Gene Deletion ◽

Genetically Modify ◽

Complex Life Cycle ◽

Mosquito Vector ◽

Parasite Line ◽

Content Type ◽

First Time

ABSTRACT Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1. IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.

Download Full-text

Purification of Components of the Translation Elongation Factor Complex of Plasmodium falciparum by Tandem Affinity Purification

Eukaryotic Cell ◽

10.1128/ec.00376-06 ◽

2007 ◽

Vol 6 (4) ◽

pp. 584-591 ◽

Cited By ~ 8

Author(s):

Sachiko Takebe ◽

William Harold Witola ◽

Bernd Schimanski ◽

Arthur Günzl ◽

Choukri Ben Mamoun

Keyword(s):

Plasmodium Falciparum ◽

Life Cycle ◽

Protein Interactions ◽

Affinity Purification ◽

Elongation Factor ◽

Tandem Affinity Purification ◽

Complex Life Cycle ◽

Spectrometric Analysis ◽

Translation Elongation Factor ◽

Translation Elongation

ABSTRACT Plasmodium falciparum is the causative agent of severe human malaria, responsible for over 2 million deaths annually. Of the 5,300 polypeptides predicted to control the parasite life cycle in mosquitoes and humans, 60% are of unknown function. A major challenge of malaria postgenomic biology is to understand how the 5,300 predicted proteins coexist and interact to perform the essential tasks that define the complex life cycle of the parasite. One approach to assign function to these proteins is by identifying their physiological partners. Here we describe the use of tandem affinity purification (TAP) and mass spectrometry for identification of native protein interactions and purification of protein complexes in P. falciparum. Transgenic parasites were generated which express the translation elongation factor PfEF-1β harboring a C-terminal PTP tag which consists of the protein C epitope, a tobacco etch virus protease cleavage site, and two protein A domains. Purification of PfEF-1β-PTP from crude extracts followed by mass spectrometric analysis revealed, in addition to the tagged protein itself, the presence of the native PfEF-1β, the G-protein PfEF-1α, and two new proteins that we named PfEF-1γ and PfEF-1δ based on their homology to other eukaryotic γ and δ translation elongation factor subunits. These data, which constitute the first application of TAP for purification of a protein complex under native conditions in P. falciparum, revealed that the translation elongation complex in this organism contains at least two subunits of PfEF-1β. The success of this approach will set the stage for a systematic analysis of protein interactions in this important human pathogen.

Download Full-text

Pre-erythrocytic antibody profiles induced by controlled human malaria infections in healthy volunteers under chloroquine prophylaxis

Scientific Reports ◽

10.1038/srep03549 ◽

2013 ◽

Vol 3 (1) ◽

Cited By ~ 55

Author(s):

Philip L. Felgner ◽

Meta Roestenberg ◽

Li Liang ◽

Christopher Hung ◽

Aarti Jain ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Life Cycle ◽

Healthy Volunteers ◽

Protective Immunity ◽

Circumsporozoite Protein ◽

Blood Stage ◽

Asexual Blood Stage ◽

Liver Stage ◽

Human Malaria ◽

Life Cycle Stages

Abstract Complete sterile protection to Plasmodium falciparum (Pf) infection mediated by pre-erythrocytic immunity can be experimentally induced under chloroquine prophylaxis, through immunization with sporozoites from infected mosquitoes' bites (CPS protocol). To characterize the profile of CPS induced antibody (Ab) responses, we developed a proteome microarray containing 809 Pf antigens showing a distinct Ab profile with recognition of antigens expressed in pre-erythrocytic life-cycle stages. In contrast, plasma from naturally exposed semi-immune individuals from Kenya was skewed toward antibody reactivity against asexual blood stage antigens. CPS-immunized and semi-immune individuals generated antibodies against 192 and 202 Pf antigens, respectively, but only 60 antigens overlapped between the two groups. Although the number of reactive antigens varied between the CPS-immunized individuals, all volunteers reacted strongly against the pre-erythrocytic antigens circumsporozoite protein (CSP) and liver stage antigen 1 (LSA1). Well classified merozoite and erythrocytic antigens were strongly reactive in semi-immune individuals but lacking in the CPS immunized group. These data show that the antibody profile of CPS-immunized and semi-immune groups have quite distinct profiles reflecting their protective immunity; antibodies from CPS immunized individuals react strongly against pre-erythrocytic while semi-immune individuals mainly react against erythrocytic antigens.

Download Full-text

Protein Prenylation and Hsp40 in Thermotolerance of Plasmodium falciparum Malaria Parasites

mBio ◽

10.1128/mbio.00760-21 ◽

2021 ◽

Author(s):

Emily S. Mathews ◽

Andrew J. Jezewski ◽

Audrey R. Odom John

Keyword(s):

Plasmodium Falciparum ◽

Life Cycle ◽

Heat Shock ◽

Heat Shock Protein ◽

Malaria Parasite ◽

Plasmodium Falciparum Malaria ◽

Complex Life Cycle ◽

Large Temperature ◽

Protein Prenylation ◽

Content Type

During its complex life cycle, the malaria parasite survives dramatic environmental stresses, including large temperature shifts. Protein prenylation is required during asexual replication of Plasmodium falciparum , and the canonical heat shock protein 40 protein (HSP40; PF3D7_1437900) is posttranslationally modified with a 15-carbon farnesyl isoprenyl group.

Download Full-text

Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei

Nucleic Acids Research ◽

10.1093/nar/gkaa641 ◽

2020 ◽

Vol 48 (15) ◽

pp. 8704-8723

Author(s):

Joseph T Smith Jr. ◽

Eva Doleželová ◽

Brianna Tylec ◽

Jonathan E Bard ◽

Runpu Chen ◽

...

Keyword(s):

Life Cycle ◽

Trypanosoma Brucei ◽

High Throughput Sequencing ◽

Environmental Changes ◽

Developmental Regulation ◽

Complex Life Cycle ◽

Mrna Levels ◽

Developmentally Regulated ◽

Life Cycle Stages

Abstract Trypanosoma brucei is a parasitic protozoan that undergoes a complex life cycle involving insect and mammalian hosts that present dramatically different nutritional environments. Mitochondrial metabolism and gene expression are highly regulated to accommodate these environmental changes, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation. Here, we use high throughput sequencing and a method for promoting life cycle changes in vitro to assess the mechanisms and timing of developmentally regulated edited mRNA expression. We show that edited CYb mRNA is downregulated in mammalian bloodstream forms (BSF) at the level of editing initiation and/or edited mRNA stability. In contrast, edited COIII mRNAs are depleted in BSF by inhibition of editing progression. We identify cell line-specific differences in the mechanisms abrogating COIII mRNA editing, including the possible utilization of terminator gRNAs that preclude the 3′ to 5′ progression of editing. By examining the developmental timing of altered mitochondrial mRNA levels, we also reveal transcript-specific developmental checkpoints in epimastigote (EMF), metacyclic (MCF), and BSF. These studies represent the first analysis of the mechanisms governing edited mRNA levels during T. brucei development and the first to interrogate U-indel editing in EMF and MCF life cycle stages.

Download Full-text