• Animals
• Malaria/transmission
• Malaria/parasitology
• Protein Biosynthesis
• Protozoan Proteins/genetics
• Protozoan Proteins/metabolism
• Plasmodium yoelii/genetics
• Plasmodium yoelii/metabolism
• Female
• Host-Parasite Interactions
• Proteomics
• Mice
• Mosquito Vectors/parasitology

• R01 AI123341 NIAID NIH HHS
• R01 AI148489 NIAID NIH HHS
• S10 OD026936 NIH HHS
• R56 AI123341 NIAID NIH HHS
• R01 GM087221 NIGMS NIH HHS
• Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases
• National Institute of General Medical Sciences
• NIH Office of the Director
• National Science Foundation
• Huck Institutes of the Life Sciences

• Plasmodium
• Differentially expressed genes (DEGs)
• Genome sequencing
• Mouse
• Polymorphism
• Proteome

• Plasmodium yoelii/genetics
• Drug Resistance/genetics
• Genome, Protozoan
• Genomics
• Animals
• Mice
• Polymorphism, Single Nucleotide
• Malaria/parasitology
• Gene Expression Regulation

• National Institute of Allergy and Infectious Diseases (NIAID)

• Plasmodium
• live attenuated parasite
• liver stage
• malaria
• sporozoite
• stearoyl‐CoA Δ9‐desaturase
• vaccine

• Plasmodium berghei/genetics
• Plasmodium berghei/growth & development
• Plasmodium berghei/metabolism
• Plasmodium berghei/enzymology
• Animals
• Mice
• Organelle Biogenesis
• Liver/parasitology
• Merozoites/growth & development
• Merozoites/metabolism
• Malaria/parasitology
• Stearoyl-CoA Desaturase/metabolism
• Stearoyl-CoA Desaturase/genetics
• Sporozoites/growth & development
• Sporozoites/metabolism
• Protozoan Proteins/metabolism
• Protozoan Proteins/genetics
• Anopheles/parasitology
• Female
• Endoplasmic Reticulum/metabolism

• BT/RLF/Re-entry/20/2012 Department of Biotechnology, Ministry of Science and Technology, India
• CRG/2022/003848 Science and Engineering Research Board

• MD simulation
• O-fucosyltransferase
• TSR domain
• glycosylation
• plasmodium
• protein stability

• Animals
• Mice
• Fucosyltransferases/genetics
• Fucosyltransferases/metabolism
• Plasmodium berghei/genetics
• Sporozoites
• Protozoan Proteins/metabolism
• Hepatocytes/parasitology

• DIA Proteomics
• Malaria Transmission
• Proximity Proteomics
• RNA-seq
• Translational Repression

• R01 AI123341 NIAID NIH HHS
• R01 AI148489 NIAID NIH HHS
• S10 OD026936 NIH HHS
• R56 AI123341 NIAID NIH HHS
• R01 GM087221 NIGMS NIH HHS

• batch correction
• corss-speices
• multi-omics
• single-cell
• spatial transcriptomics

• 2021ZD0200403 Social Trends Institute
• 62333018 National Science Foundation of China
• 2023Z226 Key Research and Development Program of Ningbo City
• 2021AB20147 Key Project of Science and Technology of Guangxi
• 2021 AC19354 Guangxi Science and Technology Base and Talents Special Project
• Guangxi Key Lab of Human-machine Interaction and Intelligent Decision, Guangxi Academy Sciences

• Plasmodium yoelii 17XNL
• gene models
• genome assembly
• long-read sequencing
• malaria

• plasmodium yoelii 17xnl
• malaria
• genome assembly
• gene model

• Plasmodium
• cell division
• malaria
• molecular screens
• sexual development

• Animals
• Anopheles/parasitology
• Female
• Malaria/parasitology
• Mosquito Vectors
• Parasites
• Plasmodium berghei/genetics

• G0900109 Medical Research Council
• FC001097 Wellcome Trust
• BB/N017609/1 Biotechnology and Biological Sciences Research Council
• G0900278 Medical Research Council
• MR/K011782/1 Medical Research Council
• Cancer Research UK

Plasmodium vivax sporozoites reside in the salivary glands of a mosquito before infecting a human host and causing malaria. Previous transcriptome-wide studies in populations of these parasite forms were limited in their ability to elucidate cell-to-cell variation, thereby masking cellular states potentially important in understanding malaria transmission outcomes.

In this study, we performed transcription profiling on 9,947 P. vivax sporozoites to assess the extent to which they differ at single-cell resolution. We show that sporozoites residing in the mosquito’s salivary glands exist in distinct developmental states, as defined by their transcriptomic signatures. Additionally, relative to P. falciparum, P. vivax displays overlapping and unique gene usage patterns, highlighting conserved and species-specific gene programs. Notably, distinguishing P. vivax from P. falciparum were a subset of P. vivax sporozoites expressing genes associated with translational regulation and repression. Finally, our comparison of single-cell transcriptomic data from P. vivax sporozoite and erythrocytic forms reveals gene usage patterns unique to sporozoites.

In defining the transcriptomic signatures of individual P. vivax sporozoites, our work provides new insights into the factors driving their developmental trajectory and lays the groundwork for a more comprehensive P. vivax cell atlas.

Plasmodium vivax is the second most common cause of malaria worldwide. It is particularly challenging for malaria elimination as it forms both active blood-stage infections, as well as asymptomatic liver-stage infections that can persist for extended periods of time. The activation of persister forms in the liver (hypnozoites) are responsible for relapsing infections occurring weeks or months following primary infection via a mosquito bite. How P. vivax persists in the liver remains a major gap in understanding of this organism. It has been hypothesized that there is pre-programming of the infectious sporozoite while it is in the salivary-glands that determines if the cell’s fate once in the liver is to progress towards immediate liver stage development or persist for long-periods as a hypnozoite. The aim of this study was to see if such differences were distinguishable at the transcript level in salivary-gland sporozoites. While we found significant variation amongst sporozoites, we did not find clear evidence that they are transcriptionally pre-programmed as has been suggested. Nevertheless, we highlight several intriguing patterns that appear to be P. vivax specific relative to non-relapsing species that cause malaria prompting further investigation.

• Animals
• Anopheles/genetics
• Anopheles/parasitology
• Humans
• Malaria/parasitology
• Malaria, Falciparum
• Malaria, Vivax/parasitology
• Plasmodium vivax/genetics
• Sequence Analysis, RNA
• Sporozoites/genetics
• Transcriptome

• U19 AI129392 NIAID NIH HHS
• Agence Nationale de la Recherche
• National Health and Medical Research Council
• National Institute of Allergy and Infectious Diseases

During transmission of malaria‐causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here, we show that the membrane‐associated protein, concavin, is important for the maintenance of the Plasmodium sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin‐GFP localizes at the cytoplasmic periphery and concavin(−) sporozoites progressively round up upon entry of salivary glands. Rounded concavin(−) sporozoites fail to pass through the narrow salivary ducts and are rarely ejected by mosquitoes, while normally shaped concavin(−) sporozoites are transmitted. Strikingly, motile concavin(−) sporozoites disintegrate while migrating through the skin leading to parasite arrest or death and decreased transmission efficiency. Collectively, we suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. Interfering with cell shape maintenance pathways might hence provide a new strategy to prevent a malaria infection.

Clostridium butyricum has been widely considered an antibiotic substitute in recent years. It can promote growth performance, improve the immune response and enhance the intestinal barrier function of the host. In the present study, 1-d-old Arbor Acres (AA) broilers were fed C. butyricum (1 × 10⁹ cfu/kg) for 28 d. The transcriptomic characteristics of epithelial cells of the cecal mucosa were determined by RNA-sequence, and the cecal microbiota composition was explored by 16S ribosomal RNA gene sequencing. The changes in the intestinal mucosa of broilers were then analyzed by tissue staining. Gene Ontology (GO) annotations identified substance transport and processes and pathways that might participate in intestinal development and cell viability. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the differentially expressed genes are involved in numerous pathways related to amino acid and vitamin metabolism and antioxidant and defensive functions, among others. The relative expression of some genes associated with intestinal barrier function (claudins 2, 15, 19, and 23, tight junction proteins 1, 2, and 3 and mucin 1) was significantly increased in the treatment group (P < 0.05 or P < 0.01). Moreover, the proportion of Firmicutes was higher in the C. butyricum-treated group, whereas the proportion of Proteobacteria was higher in the control group. At the genus level, the relative abundances of Butyricicoccus and Lactobacillus, among other bacteria, were increased after C. butyricum supplementation. The tissue staining analysis showed that the cecal mucosa of broilers was significantly ameliorated after the addition of C. butyricum (P < 0.05 or P < 0.01). These results showed that dietary supplementation with C. butyricum can enhance the antioxidant capacity, mucosal barrier function, and stabilize the cecal microbiota, resulting in improving the growth performance.

• 16S ribosomal RNA
• Broiler
• Clostridium butyricum
• Intestinal barrier
• RNA-sequence

Productive transmission of malaria parasites hinges upon the execution of key transcriptional and posttranscriptional regulatory events. While much is now known about how specific transcription factors activate or repress sexual commitment programs, far less is known about the production of a preferred mRNA homeostasis following commitment and through the host-to-vector transmission event. Here, we show that in Plasmodium parasites, the NOT1 scaffold protein of the CAF1/CCR4/Not complex is duplicated, and one paralogue is dedicated for essential transmission functions. Moreover, this NOT1-G paralogue is central to the sex-specific functions previously associated with its interacting partners, as deletion of not1-g in Plasmodium yoelii leads to a comparable or complete arrest phenotype for both male and female parasites. We show that, consistent with its role in other eukaryotes, PyNOT1-G localizes to cytosolic puncta throughout much of the Plasmodium life cycle. PyNOT1-G is essential to both the complete maturation of male gametes and to the continued development of the fertilized zygote originating from female parasites. Comparative transcriptomics of wild-type and pynot1-g⁻ parasites shows that loss of PyNOT1-G leads to transcript dysregulation preceding and during gametocytogenesis and shows that PyNOT1-G acts to preserve mRNAs that are critical to sexual and early mosquito stage development. Finally, we demonstrate that the tristetraprolin (TTP)-binding domain, which acts as the typical organization platform for RNA decay (TTP) and RNA preservation (ELAV/HuR) factors is dispensable for PyNOT1-G’s essential blood stage functions but impacts host-to-vector transmission. Together, we conclude that a NOT1-G paralogue in Plasmodium fulfills the complex transmission requirements of both male and female parasites.

Malaria parasites face two bottlenecks in their life cycle: their two transmission events. This study shows that Plasmodium has taken the unorthodox approach of duplicating the gene for the NOT1 RNA regulatory scaffold protein, allowing it to dedicate one paralog to functions that are essential for transmission from mammalian hosts to the mosquito vector.

• Plasmodium falciparum
• apicoplast
• fosmidomycin
• genome-scale metabolic model
• metabolic modeling
• metabolomics
• prenylation
• transcriptomics

•

Apicoplast LipB deletion leads to changed antioxidant expression that precedes and coincides with accelerated differentiation.

Malaria is still one of the most important global infectious diseases. Emergence of drug resistance and a shortage of new efficient antimalarials continue to hamper a malaria eradication agenda. Malaria parasites are highly sensitive to changes in the redox environment. Understanding the mechanisms regulating parasite redox could contribute to the design of new drugs. Malaria parasites have a complex network of redox regulatory systems housed in their cytosol, in their mitochondrion and in their plastid (apicoplast). While the roles of enzymes of the thioredoxin and glutathione pathways in parasite survival have been explored, the antioxidant role of α-lipoic acid (LA) produced in the apicoplast has not been tested. To take a first step in teasing a putative role of LA in redox regulation, we analysed a mutant Plasmodium falciparum (3D7 strain) lacking the apicoplast lipoic acid protein ligase B (lipB) known to be depleted of LA. Our results showed a change in expression of redox regulators in the apicoplast and the cytosol. We further detected a change in parasite central carbon metabolism, with lipB deletion resulting in changes to glycolysis and tricarboxylic acid cycle activity. Further, in another Plasmodium cell line (NF54), deletion of lipB impacted development in the mosquito, preventing the detection of infectious sporozoite stages. While it is not clear at this point if the observed phenotypes are linked, these findings flag LA biosynthesis as an important subject for further study in the context of redox regulation in asexual stages, and point to LipB as a potential target for the development of new transmission drugs.

• Antioxidant
• Apicoplast
• Lipoic acid
• Malaria
• Metabolism
• Plasmodium falciparum
• Redox
• Sporogony

Colonization of the mosquito host by Plasmodium parasites is achieved by sexually differentiated gametocytes. Gametocytogenesis, gamete formation and fertilization are tightly regulated processes, and translational repression is a major regulatory mechanism for stage conversion. Here, we present a characterization of a Plasmodium berghei RNA binding protein, UIS12, that contains two conserved eukaryotic RNA recognition motifs (RRM). Targeted gene deletion resulted in viable parasites that replicate normally during blood infection, but form fewer gametocytes. Upon transmission to Anopheles stephensi mosquitoes, both numbers and size of midgut-associated oocysts were reduced and their development stopped at an early time point. As a consequence, no salivary gland sporozoites were formed indicative of a complete life cycle arrest in the mosquito vector. Comparative transcript profiling in mutant and wild-type infected red blood cells revealed a decrease in transcript abundance of mRNAs coding for signature gamete-, ookinete-, and oocyst-specific proteins in uis12(-) parasites. Together, our findings indicate multiple roles for UIS12 in regulation of gene expression after blood infection in good agreement with the pleiotropic defects that terminate successful sporogony and onward transmission to a new vertebrate host.

• Plasmodium
• RNA
• RNA binding protein (RBP)
• gametocyte
• malaria
• oocyst
• translational regulation

• Plasmodium
• gene regulation
• malaria
• quiescence
• sporozoite
• transcription
• translational repression

• Neospora caninum
• Puf protein
• cyst formation
• tachyzoites replication

The regulation of gene expression in trypanosomatids occurs mainly at the post-transcriptional level. In the case of Trypanosoma cruzi, the characterization of messenger ribonucleoprotein (mRNP) particles has allowed the identification of several classes of RNA binding proteins (RBPs), as well as non-canonical RBPs, associated with mRNA molecules. The protein composition of the mRNPs as well as the localization and functionality of the mRNAs depend on their associated proteins. mRNPs can also be organized into larger complexes forming RNA granules, which function as stress granules or P-bodies depending on the associated proteins. The fate of mRNAs in the cell, and consequently the genes expressed, depends on the set of proteins associated with the messenger molecule. These proteins allow the coordinated expression of mRNAs encoding proteins that are related in function, resulting in the formation of post-transcriptional operons. However, the puzzle posed by the combinatorial association of sets of RBPs with mRNAs and how this relates to the expressed genes remain to be elucidated. One important tool in this endeavor is the use of the CRISPR/CAS system to delete genes encoding RBPs, allowing the evaluation of their effect on the formation of mRNP complexes and associated mRNAs in the different compartments of the translation machinery. Accordingly, we recently established this methodology for T. cruzi and deleted the genes encoding RBPs containing zinc finger domains. In this manuscript, we will discuss the data obtained and the potential of the CRISPR/CAS methodology to unveil the role of RBPs in T. cruzi gene expression regulation.

• CRISPR/CAS
• RNA binding proteins
• RNA granules
• Trypanosoma cruzi
• gene expression regulation
• zinc finger protein

• Animals
• Humans
• Malaria/metabolism
• Malaria/transmission
• Plasmodium/metabolism
• Plasmodium/pathogenicity
• Protozoan Proteins/metabolism
• RNA, Protozoan/metabolism

• R01 AI123341 NIAID NIH HHS

Plasmodium sporozoites are transmitted from infected mosquitoes to mammals, and must navigate the host skin and vasculature to infect the liver. This journey requires distinct proteomes. Here, we report the dynamic transcriptomes and proteomes of both oocyst sporozoites and salivary gland sporozoites in both rodent-infectious Plasmodium yoelii parasites and human-infectious Plasmodium falciparum parasites. The data robustly define mRNAs and proteins that are upregulated in oocyst sporozoites (UOS) or upregulated in infectious sporozoites (UIS) within the salivary glands, including many that are essential for sporozoite functions in the vector and host. Moreover, we find that malaria parasites use two overlapping, extensive, and independent programs of translational repression across sporozoite maturation to temporally regulate protein expression. Together with gene-specific validation experiments, these data indicate that two waves of translational repression are implemented and relieved at different times during sporozoite maturation, migration and infection, thus promoting their successful development and vector-to-host transition.

Here, the authors report transcriptomes and proteomes of oocyst sporozoite and salivary gland sporozoite stages in rodent-infectious Plasmodium yoelii parasites and human infectious Plasmodium falciparum parasites and define two waves of translational repression during sporozoite maturation.

• Life cycle
• Malaria
• Merosome
• Plasmodium liver stage
• RNA-seq
• Transcriptome

• Erythrocytes/parasitology
• Gene Expression Profiling
• Gene Expression Regulation
• Genome, Protozoan
• Hepatocytes/parasitology
• Humans
• Life Cycle Stages
• Liver/parasitology
• Malaria/parasitology
• Merozoites/genetics
• Merozoites/growth & development
• Plasmodium berghei/genetics
• Plasmodium berghei/growth & development
• Promoter Regions, Genetic
• Protozoan Proteins/genetics
• RNA-Seq
• Sporozoites/genetics
• Sporozoites/growth & development

• 51RTPO_151032 SystemsX.ch

• ALBA
• CRISPR/Cas
• HDR
• Plasmodium
• UIS4
• gene regulation
• parasitology
• ribozyme (catalytic RNA) (RNA enzyme)
• sgRNA

• Animals
• CRISPR-Associated Protein 9/genetics
• CRISPR-Associated Protein 9/metabolism
• Clustered Regularly Interspaced Short Palindromic Repeats
• Female
• Gene Deletion
• Gene Editing/methods
• Genetic Vectors
• Malaria/parasitology
• Malaria/veterinary
• Mice
• Plasmids
• Plasmodium yoelii/genetics
• Plasmodium yoelii/isolation & purification
• RNA, Catalytic
• Rodent Diseases/parasitology

• R01 AI123341 NIAID NIH HHS
• R21 AI130692 NIAID NIH HHS
• HHS NIH National Institute of Allergy and Infectious Diseases (NIAID)

• CHiP-Seq
• Dormancy
• Hypnozoite
• Plasmodium vivax
• RNA-seq
• Sporozoite
• Transcriptome

• Malaria
• Microsampling
• Plasmodium
• Rodent malaria parasites
• Time-series
• Transcriptomics

• Animals
• Culicidae/metabolism
• Exoribonucleases
• Gametogenesis/physiology
• Gene Expression Regulation
• Homeodomain Proteins
• Male
• Mice
• Mosquito Vectors
• Plasmodium/genetics
• Plasmodium falciparum/genetics
• Plasmodium falciparum/metabolism
• Proteins
• RNA, Messenger/genetics
• Receptors, CCR4/metabolism
• Repressor Proteins
• Ribonucleases
• Transcription Factors/metabolism
• Transcriptional Activation

• K22 AI101039 NIAID NIH HHS
• R01 AI123341 NIAID NIH HHS
• F32 AI112271 NIAID NIH HHS
• F32 AI010139 NIAID NIH HHS
• R01 AI130171 NIAID NIH HHS
• R01 AI094973 NIAID NIH HHS
• National Institute of Allergy and Infectious Diseases

• Animals
• Anopheles/parasitology
• Gene Expression Profiling
• Host-Pathogen Interactions/genetics
• Humans
• Insect Vectors/parasitology
• Malaria/parasitology
• Malaria, Vivax/parasitology
• Mosquito Vectors/genetics
• Parasites
• Plasmodium vivax/genetics
• Plasmodium vivax/pathogenicity
• Plasmodium vivax/physiology
• Salivary Glands/parasitology
• Sporozoites/genetics
• Sporozoites/pathogenicity
• Sporozoites/physiology

• Bcl-6
• CD4 T cell
• OX40
• T follicular helper cell
• T-bet
• memory
• plasmodium
• type 1 T helper cell

• Plasmodium
• Puf protein
• RNA-binding protein
• Ribosomal biogenesis
• Translational regulation

Malaria remains one of the great global health problems. The parasite that causes malaria (Plasmodium genus) relies upon exquisite control of its transmission between vertebrate hosts and mosquitoes. One crucial way that it does so is by proactively producing mRNAs needed to establish the new infection but by silencing and storing them until they are needed. One key protein in this process of translational repression in model eukaryotes is poly(A)-binding protein (PABP). Here we have shown that Plasmodium yoelii utilizes both a nuclear PABP and a cytosolic PABP, both of which bind specifically to polyadenylated RNA sequences. Moreover, we find that the cytosolic PABP forms chains in vitro, consistent with its appreciated role in coating the poly(A) tails of mRNA. Finally, we have also verified that, surprisingly, the cytosolic PABP is found on the surface of Plasmodium sporozoites. Taking the data together, we propose that Plasmodium utilizes a more metazoan-like strategy for RNA metabolism using specialized PABPs.

Malaria is a devastating illness that causes approximately 500,000 deaths annually. The malaria-causing parasite (Plasmodium genus) uses the process of translational repression to regulate its growth, development, and transmission. As poly(A)-binding proteins (PABP) have been identified as critical components of RNA metabolism and translational repression in model eukaryotes and in Plasmodium, we have identified and investigated two PABPs in Plasmodium yoelii, PyPABP1 and PyPABP2. In contrast to most single-celled eukaryotes, Plasmodium closely resembles metazoans and encodes both a nuclear PABP and a cytosolic PABP; here, we provide multiple lines of evidence in support of this observation. The conserved domain architectures of PyPABP1 and PyPABP2 resemble those of yeast and metazoans, while multiple independent binding assays demonstrated their ability to bind very strongly and specifically to poly(A) sequences. Interestingly, we also observed that purified PyPABP1 forms homopolymeric chains despite exhaustive RNase treatment in vitro. Finally, we show by indirect immunofluorescence assays (IFAs) that PyPABP1 and PyPABP2 are cytoplasm- and nucleus-associated PABPs during the blood stages of the life cycle. Surprisingly, however, PyPABP1 was instead observed to also be localized on the surface of transmitted salivary gland sporozoites and to be deposited in trails when parasites glide on a substrate. This is the third RNA-binding protein verified to be found on the sporozoite surface, and the data may point to an unappreciated RNA-centered interface between the host and parasite.

IMPORTANCE Malaria remains one of the great global health problems. The parasite that causes malaria (Plasmodium genus) relies upon exquisite control of its transmission between vertebrate hosts and mosquitoes. One crucial way that it does so is by proactively producing mRNAs needed to establish the new infection but by silencing and storing them until they are needed. One key protein in this process of translational repression in model eukaryotes is poly(A)-binding protein (PABP). Here we have shown that Plasmodium yoelii utilizes both a nuclear PABP and a cytosolic PABP, both of which bind specifically to polyadenylated RNA sequences. Moreover, we find that the cytosolic PABP forms chains in vitro, consistent with its appreciated role in coating the poly(A) tails of mRNA. Finally, we have also verified that, surprisingly, the cytosolic PABP is found on the surface of Plasmodium sporozoites. Taking the data together, we propose that Plasmodium utilizes a more metazoan-like strategy for RNA metabolism using specialized PABPs.

• PABP
• Plasmodium
• RNA metabolism
• electron microscopy
• poly(A)-binding protein
• surface proteins
• yoelii

Despite global eradication efforts over the past century, malaria remains a devastating public health burden, causing almost half a million deaths annually (WHO, 2016). A detailed understanding of the mechanisms that control malaria infection has been hindered by technical challenges of studying a complex parasite life cycle in multiple hosts. While many interventions targeting the parasite have been implemented, the complex biology of Plasmodium poses a major challenge, and must be addressed to enable eradication. New approaches for elucidating key host-parasite interactions, and predicting how the parasite will respond in a variety of biological settings, could dramatically enhance the efficacy and longevity of intervention strategies. The field of systems biology has developed methodologies and principles that are well poised to meet these challenges. In this review, we focus our attention on the Liver Stage of the Plasmodium lifecycle and issue a “call to arms” for using systems biology approaches to forge a new era in malaria research. These approaches will reveal insights into the complex interplay between host and pathogen, and could ultimately lead to novel intervention strategies that contribute to malaria eradication.

• computational modeling
• liver
• malaria
• omics-technologies
• plasmodium
• systems biology

• Plasmodium
• Toxoplasma
• eIF2
• latency
• translational control

To capture the transcriptional dynamics within proliferating cells, methods to differentiate nascent transcription from preexisting mRNAs are desired. One approach is to label newly synthesized mRNA transcripts in vivo through the incorporation of modified pyrimidines. However, the human malaria parasite, Plasmodium falciparum, is incapable of pyrimidine salvage for mRNA biogenesis. To capture cellular mRNA dynamics during Plasmodium development, we engineered parasites that can salvage pyrimidines through the expression of a single bifunctional yeast fusion gene, cytosine deaminase/uracil phosphoribosyltransferase (FCU). We show that expression of FCU allows for the direct incorporation of thiol-modified pyrimidines into nascent mRNAs. Using developmental stage-specific promoters to express FCU-GFP enables the biosynthetic capture and in-depth analysis of mRNA dynamics from subpopulations of cells undergoing differentiation. We demonstrate the utility of this method by examining the transcriptional dynamics of the sexual gametocyte stage transition, a process that is essential to malaria transmission between hosts. Using the pfs16 gametocyte-specific promoter to express FCU-GFP in 3D7 parasites, we found that sexual stage commitment is governed by transcriptional reprogramming and stabilization of a subset of essential gametocyte transcripts. We also measured mRNA dynamics in F12 gametocyte-deficient parasites and demonstrate that the transcriptional program required for sexual commitment and maturation is initiated but likely aborted due to the absence of the PfAP2-G transcriptional regulator and a lack of gametocyte-specific mRNA stabilization. Biosynthetic labeling of Plasmodium mRNAs is incredibly versatile, can be used to measure transcriptional dynamics at any stage of parasite development, and will allow for future applications to comprehensively measure RNA-protein interactions in the malaria parasite.

P. falciparum phenotypic plasticity is linked to the variant expression of clonal multigene families such as the var genes. We have examined changes in transcription and histone modifications that occur during sporogonic development of P. falciparum in the mosquito host. All var genes are silenced or transcribed at low levels in blood stages (gametocyte/ring) of the parasite in the human host. After infection of mosquitoes, a single var gene is selected for expression in the oocyst, and transcription of this gene increases dramatically in the sporozoite. The same PF3D7_1255200 var gene was activated in 4 different experimental infections. Transcription of this var gene during parasite development in the mosquito correlates with the presence of low levels of H3K9me3 at the binding site for the PF3D7_1466400 AP2 transcription factor. This chromatin state in the sporozoite also correlates with the expression of an antisense long non-coding RNA (lncRNA) that has previously been shown to promote var gene transcription during the intraerythrocytic cycle in vitro. Expression of both the sense protein-coding transcript and the antisense lncRNA increase dramatically in sporozoites. The findings suggest a complex process for the activation of a single particular var gene that involves AP2 transcription factors and lncRNAs.

• Gametocyte
• Plasmodium falciparum
• Puf family
• Sex ratio
• Translation regulation

Gene expression is controlled at multiple levels, including transcription, stability, translation, and degradation. Over the years, it has become apparent that Plasmodium falciparum exerts limited transcriptional control of gene expression, while at least part of Plasmodium’s genome is controlled by post-transcriptional mechanisms. To generate insights into the mechanisms that regulate gene expression at the post-transcriptional level, we undertook complementary computational, comparative genomics, and experimental approaches to identify and characterize mRNA-binding proteins (mRBPs) in P. falciparum.

Close to 1000 RNA-binding proteins are identified by hidden Markov model searches, of which mRBPs encompass a relatively large proportion of the parasite proteome as compared to other eukaryotes. Several abundant mRNA-binding domains are enriched in apicomplexan parasites, while strong depletion of mRNA-binding domains involved in RNA degradation is observed. Next, we experimentally capture 199 proteins that interact with mRNA during the blood stages, 64 of which with high confidence. These captured mRBPs show a significant overlap with the in silico identified candidate RBPs (p < 0.0001). Among the experimentally validated mRBPs are many known translational regulators active in other stages of the parasite’s life cycle, such as DOZI, CITH, PfCELF2, Musashi, and PfAlba1–4. Finally, we also detect several proteins with an RNA-binding domain abundant in Apicomplexans (RAP domain) that is almost exclusively found in apicomplexan parasites.

Collectively, our results provide the most complete comparative genomics and experimental analysis of mRBPs in P. falciparum. A better understanding of these regulatory proteins will not only give insight into the intricate parasite life cycle but may also provide targets for novel therapeutic strategies.

The online version of this article (doi:10.1186/s13059-016-1014-0) contains supplementary material, which is available to authorized users.

• Gene expression
• Post-transcriptional regulation
• Protein domains
• RNA-binding proteins
• Translation

Malaria parasite infection is initiated by the mosquito-transmitted sporozoite stage, a highly motile invasive cell that targets hepatocytes in the liver for infection. A promising approach to developing a malaria vaccine is the use of proteins located on the sporozoite surface as antigens to elicit humoral immune responses that prevent the establishment of infection. Very little of the P. falciparum genome has been considered as potential vaccine targets, and candidate vaccines have been almost exclusively based on single antigens, generating the need for novel target identification. The most advanced malaria vaccine to date, RTS,S, a subunit vaccine consisting of a portion of the major surface protein circumsporozoite protein (CSP), conferred limited protection in Phase III trials, falling short of community-established vaccine efficacy goals. In striking contrast to the limited protection seen in current vaccine trials, sterilizing immunity can be achieved by immunization with radiation-attenuated sporozoites, suggesting that more potent protection may be achievable with a multivalent protein vaccine. Here, we provide the most comprehensive analysis to date of proteins located on the surface of or secreted by Plasmodium falciparum salivary gland sporozoites. We used chemical labeling to isolate surface-exposed proteins on sporozoites and identified these proteins by mass spectrometry. We validated several of these targets and also provide evidence that components of the inner membrane complex are in fact surface-exposed and accessible to antibodies in live sporozoites. Finally, our mass spectrometry data provide the first direct evidence that the Plasmodium surface proteins CSP and TRAP are glycosylated in sporozoites, a finding that could impact the selection of vaccine antigens.

Malaria remains one of the most important infectious diseases in the world, responsible for an estimated 500 million new cases and 600,000 deaths annually. The etiologic agents of the disease are protozoan parasites of the genus Plasmodium that have a complex cycle between mosquito and mammalian hosts. Though all clinical symptoms are attributable to the blood stages, it is only by attacking the transmission stages that we can make an impact on the economic and health burdens of malaria. Infection is initiated when mosquitoes inoculate sporozoites into the skin as they probe for blood. Sporozoites must locate blood vessels and enter the circulation to reach the liver where they invade and grow in hepatocytes. The inoculum is low and these early stages of infection are asymptomatic. Though the small amounts of material available for study has made large scale -omics studies difficult, killing the parasite at this stage would prevent infection and block downstream transmission to mosquitoes, thus preventing spread of disease. Here we use state-of-the-art biochemistry tools to identify the proteins on the sporozoite surface and find that two of the most studied proteins, CSP and TRAP, have post-translational modifications. These studies will aid investigations into the novel biology of sporozoites and importantly, significantly expand the pool of potential vaccine candidates.

• Animals
• Culicidae
• Fluorescent Antibody Technique
• Glycosylation
• Malaria, Falciparum/metabolism
• Mass Spectrometry
• Membrane Proteins/analysis
• Membrane Proteins/metabolism
• Organisms, Genetically Modified
• Proteomics/methods
• Protozoan Proteins/analysis
• Protozoan Proteins/metabolism
• Sporozoites/chemistry
• Sporozoites/metabolism

• R01 GM087221 NIGMS NIH HHS
• R01 AI056840 NIAID NIH HHS
• P50 GM076547 NIGMS NIH HHS
• S10 RR027584 NCRR NIH HHS
• R01 AI095686 NIAID NIH HHS
• K25 AI119229 NIAID NIH HHS
• K22 AI101039 NIAID NIH HHS
• Bill and Melinda Gates Foundation
• National Institute of Allergy and Infectious Diseases
• National Science Foundation
• National Institute of General Medical Sciences

RNA-binding proteins (RBPs) are key regulators of gene expression. There are several distinct families of RBPs and they are involved in the cellular response to environmental changes, cell differentiation and cell death. The RBPs can differentially combine with RNA molecules and form ribonucleoprotein (RNP) complexes, defining the function and fate of RNA molecules in the cell. RBPs display diverse domains that allow them to be categorized into distinct families. They play important roles in the cellular response to physiological stress, in cell differentiation, and, it is believed, in the cellular localization of certain mRNAs. In several protozoa, a physiological stress (nutritional, temperature or pH) triggers differentiation to a distinct developmental stage. Most of the RBPs characterized in protozoa arise from trypanosomatids. In these protozoa gene expression regulation is mostly post-transcriptional, which suggests that some RBPs might display regulatory functions distinct from those described for other eukaryotes. mRNA stability can be altered as a response to stress. Transcripts are sequestered to RNA granules that ultimately modulate their availability to the translation machinery, storage or degradation, depending on the associated proteins. These aggregates of mRNPs containing mRNAs that are not being translated colocalize in cytoplasmic foci, and their numbers and size vary according to cell conditions such as oxidative stress, nutritional status and treatment with drugs that inhibit translation.

• Gene expression regulation
• RNA-binding proteins
• RNA-protein complexes
• RNA granules
• Protozoa
• Stress and cell differentiation

• Animals
• Gene Expression Regulation
• Host-Parasite Interactions
• Immunoprecipitation
• Plasmodium berghei/metabolism
• Protozoan Proteins/genetics
• Protozoan Proteins/metabolism
• RNA-Binding Proteins/metabolism
• Salivary Glands/metabolism
• Sporozoites/metabolism

The rodent malaria parasite Plasmodium yoelii is an important animal model for studying host-parasite interaction and molecular basis of malaria pathogenesis. Although a draft genome of P. yoeliiyoelii YM is available, and RNA sequencing (RNA-seq) data for several rodent malaria species (RMP) were reported recently, variations in coding regions and structure of mRNA transcript are likely present between different parasite strains or subspecies. Sequencing of cDNA libraries from additional parasite strains/subspecies will help improve the gene models and genome annotation.

Here two directional cDNA libraries from mixed blood stages of a subspecies of P. yoelii (P. y. nigeriensis NSM) with or without mefloquine (MQ) treatment were sequenced, and the sequence reads were compared to the genome and cDNA sequences of P. y. yoelii YM in public databases to investigate single nucleotide polymorphisms (SNPs) in coding regions, variations in intron–exon structure and differential splicing between P. yoelii subspecies, and variations in gene expression under MQ pressure.

Approximately 56 million of 100 bp paired-end reads were obtained, providing an average of ~225-fold coverage for the coding regions. Comparison of the sequence reads to the YM genome revealed introns in 5′ and 3′ untranslated regions (UTRs), altered intron/exon boundaries, alternative splicing, overlapping sense-antisense reads, and potentially new transcripts. Interestingly, comparison of the NSM RNA-seq reads obtained here with those of YM discovered differentially spliced introns; e.g., spliced introns in one subspecies but not the other. Alignment of the NSM cDNA sequences to the YM genome sequence also identified ~84,000 SNPs between the two parasites.

The discoveries of UTR introns and differentially spliced introns between P. yoelii subspecies raise interesting questions on the potential role of these introns in regulating gene expression and evolution of malaria parasites.

The online version of this article (doi:10.1186/s12936-015-1081-9) contains supplementary material, which is available to authorized users.