scholarly journals CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Jessica M. Bryant ◽  
Clément Regnault ◽  
Christine Scheidig-Benatar ◽  
Sebastian Baumgarten ◽  
Julien Guizetti ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Transcriptional Regulation ◽  
Gene Family ◽  
Genome Editing ◽  
Immune Evasion ◽  
Antigenic Variation ◽  
Surface Proteins ◽  
Functional Study ◽  
Monoallelic Expression ◽  
Immune Evasion Mechanism

ABSTRACT Plasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the var promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family. IMPORTANCE Plasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes—the intron—in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression. IMPORTANCE Plasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes—the intron—in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression.

Identification of avir-orthologous immune evasion gene family from primate malaria parasites

Parasitology ◽  
2014 ◽  
Vol 141 (5) ◽  
pp. 641-645 ◽  
Author(s):  
SURENDRA KUMAR PRAJAPATI ◽  
OM PRAKASH SINGH
Keyword(s):  
Gene Family ◽  
Immune Evasion ◽  
Experimental Approach ◽  
Antigenic Variation ◽  
Surface Proteins ◽  
Malaria Parasites ◽  
Sequence Identity ◽  
Plasmodium Cynomolgi ◽  
Evolution Of Virulence ◽  
Insight Into

SUMMARYThe immune evasion gene family of malaria parasites encodes variant surface proteins that are expressed at the surface of infected erythrocytes and help the parasite in evading the host immune response by means of antigenic variation. The identification ofPlasmodium vivax virorthologous immune evasion gene family from primate malaria parasites would provide new insight into the evolution of virulence and pathogenesis. Threevirsubfamilies viz.vir-B, vir-Dandvir-Gwere successfully PCR amplified from primate malaria parasites, cloned and sequenced. DNA sequence analysis confirmed orthologues ofvir-Dsubfamily inPlasmodium cynomolgi, Plasmodium simium, Plasmodium simiovaleandPlasmodium fieldi. The identifiedvir-Dorthologues are 1–9 distinct members of the immune evasion gene family which have 68–83% sequence identity withvir-Dand 71·2–98·5% sequence identity within the members identified from primate malaria parasites. The absence of othervirsubfamilies among primate malaria parasites reflects the limitations in the experimental approach. This study clearly identified the presence ofvir-Dlike sequences in four species ofPlasmodiuminfecting primates that would be useful in understanding the evolution of virulence in malaria parasites.

scholarly journals CRISPR Interference of a Clonally Variant GC-Rich Noncoding RNA Family Leads to General Repression of var Genes in Plasmodium falciparum

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna Barcons-Simon ◽  
Carlos Cordon-Obras ◽  
Julien Guizetti ◽  
Jessica M. Bryant ◽  
Artur Scherf
Keyword(s):  
Plasmodium Falciparum ◽  
Gene Family ◽  
Immune Evasion ◽  
Noncoding Rna ◽  
Malaria Parasite ◽  
Antigenic Variation ◽  
Activation Mechanism ◽  
Regulation Mechanism ◽  
Content Type ◽  
Crispr Interference

ABSTRACT The human malaria parasite Plasmodium falciparum uses mutually exclusive expression of the PfEMP1-encoding var gene family to evade the host immune system. Despite progress in the molecular understanding of the default silencing mechanism, the activation mechanism of the uniquely expressed var member remains elusive. A GC-rich noncoding RNA (ncRNA) gene family has coevolved with Plasmodium species that express var genes. Here, we show that this ncRNA family is transcribed in a clonally variant manner, with predominant transcription of a single member occurring when the ncRNA is located adjacent to and upstream of an active var gene. We developed a specific CRISPR interference (CRISPRi) strategy that allowed for the transcriptional repression of all GC-rich members. A lack of GC-rich ncRNA transcription led to the downregulation of the entire var gene family in ring-stage parasites. Strikingly, in mature blood-stage parasites, the GC-rich ncRNA CRISPRi affected the transcription patterns of other clonally variant gene families, including the downregulation of all Pfmc-2TM members. We provide evidence for the key role of GC-rich ncRNA transcription in var gene activation and discovered a molecular link between the transcriptional control of various clonally variant multigene families involved in parasite virulence. This work opens new avenues for elucidating the molecular processes that control immune evasion and pathogenesis in P. falciparum. IMPORTANCE Plasmodium falciparum is the deadliest malaria parasite species, accounting for the vast majority of disease cases and deaths. The virulence of this parasite is reliant upon the mutually exclusive expression of cytoadherence proteins encoded by the 60-member var gene family. Antigenic variation of this multigene family serves as an immune evasion mechanism, ultimately leading to chronic infection and pathogenesis. Understanding the regulation mechanism of antigenic variation is key to developing new therapeutic and control strategies. Our study uncovers a novel layer in the epigenetic regulation of transcription of this family of virulence genes by means of a multigene-targeting CRISPR interference approach.

scholarly journals The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of plasmodium falciparum-infected erythrocytes

Cell ◽  
1995 ◽  
Vol 82 (1) ◽  
pp. 89-100 ◽  
Author(s):  
Xin-zhuan Su ◽  
Virginia M. Heatwole ◽  
Samuel P. Wertheimer ◽  
Frangoise Guinet ◽  
Jacqueline A. Herrfeldt ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Gene Family ◽  
Antigenic Variation

scholarly journals Expressed var genes are found in Plasmodium falciparum subtelomeric regions.

1997 ◽  
Vol 17 (2) ◽  
pp. 604-611 ◽  
Author(s):  
R Hernandez-Rivas ◽  
D Mattei ◽  
Y Sterkers ◽  
D S Peterson ◽  
T E Wellems ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Antigenic Variation ◽  
Protein Product ◽  
Terminal Segment ◽  
Surface Proteins ◽  
Var Genes ◽  
Active Expression ◽  
Transmembrane Sequence ◽  
High Level ◽  
Carboxy Terminal

The antigenic variation and cytoadherence of Plasmodium falciparum-infected erythrocytes are modulated by a family of variant surface proteins encoded by the var multigene family. The var genes occur on multiple chromosomes, often in clusters, and 50 to 150 genes are estimated to be present in the haploid parasite genome. Transcripts from var genes have been previously mapped to internal chromosome positions, but the generality of such assignments and the expression sites and mechanisms that control switches of var gene expression are still in early stages of investigation. Here we describe investigations of closely related var genes that occur in association with repetitive elements near the telomeres of P. falciparum chromosomes. DNA sequence analysis of one of these genes (FCR3-varT11-1) shows the characteristic two-exon structure encoding expected var features, including three variable Duffy binding-like (DBL) domains, a transmembrane sequence, and a carboxy-terminal segment thought to anchor the protein product in knobs at the surface of the parasitized erythrocyte. FCR3-varT11-1 cross-hybridizes with var genes located close to the telomeres of many other P. falciparum chromosomes, including a transcribed gene (FCR3-varT3-1) in chromosome 3 of the P. falciparum FCR3 line. The relatively high level transcription from this gene shows that the polymorphic chromosome ends of P. falciparum, which have been proposed to be transcriptionally silent, can be active expression sites for var genes. The pattern of the FCR3-varT11-1 and FCR3-varT3-1 genes are variable between different P. falciparum lines, presumably due to DNA rearrangements. Thus, recombination events in subtelomeric DNA may have a role in the expression of novel var forms.

scholarly journals Diversity and Complexity of the Large Surface Protein Family in the Compacted Genomes of Multiple Pneumocystis Species

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Liang Ma ◽  
Zehua Chen ◽  
Da Wei Huang ◽  
Ousmane H. Cissé ◽  
Jamie L. Rothenburger ◽  
...  
Keyword(s):  
Immune Evasion ◽  
Disease Transmission ◽  
Antigenic Variation ◽  
The United States ◽  
Surface Proteins ◽  
Surface Glycoprotein ◽  
Mammalian Host ◽  
Content Type ◽  
Multiple Functions ◽  
First Time

ABSTRACT Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multicopy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins and provide a detailed description of the classification, unique characteristics, and phylogenetic relatedness of five Msg families. We further describe, for the first time, the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests multiple functions for this superfamily rather than just conferring antigenic variation to allow immune evasion as previously believed. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology. IMPORTANCE Pneumocystis continues to be a major cause of disease in humans with immunodeficiency, especially those with HIV/AIDS and organ transplants, and is being seen with increasing frequency worldwide in patients treated with immunodepleting monoclonal antibodies. Annual health care associated with Pneumocystis pneumonia costs ∼$475 million dollars in the United States alone. In addition to causing overt disease in immunodeficient individuals, Pneumocystis can cause subclinical infection or colonization in healthy individuals, which may play an important role in species preservation and disease transmission. Our work sheds new light on the diversity and complexity of the msg superfamily and strongly suggests that the versatility of this superfamily reflects multiple functions, including antigenic variation to allow immune evasion and optimal adaptation to host environmental conditions to promote efficient infection and transmission. These findings are essential to consider in developing new diagnostic and therapeutic strategies.

Disruption of Plasmodium berghei merozoite surface protein 7 gene modulates parasite growth in vivo

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 394-396 ◽  
Author(s):  
Rita Tewari ◽  
Solabomi A. Ogun ◽  
Ruwani S. Gunaratne ◽  
Andrea Crisanti ◽  
Anthony A. Holder
Keyword(s):  
Plasmodium Falciparum ◽  
Gene Family ◽  
Plasmodium Berghei ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Plasmodium Yoelii ◽  
Parasite Growth ◽  
Surface Proteins ◽  
Rate Of Increase

Abstract Merozoite invasion of red blood cells is crucial to the development of the parasite that causes malaria. Merozoite surface proteins (MSPs) mediate the first interaction between parasite and erythrocyte. In Plasmodium falciparum, they include a complex of products from at least 3 genes (msp1, msp6, and msp7), one of which, msp7, is part of a gene family containing 3 and 6 adjacent members in Plasmodium yoelii and Plasmodium falciparum, respectively. We have identified and disrupted msp7 in the Plasmodium berghei gene family. The protein is expressed in schizonts and colocalizes with MSP1. The synthesis and processing of MSP1 was unaffected in the parasite with the disrupted gene (MSP7ko). Disruption of msp7 was not lethal but affected blood-stage parasite growth. MSP7ko parasites initially grew more slowly than wild-type parasites. However, when reticulocytes were prevalent, the rate of increase in parasitemia was similar, suggesting that MSP7ko parasites prefer to invade and grow within reticulocytes. (Blood. 2005;105:394-396)

scholarly journals Telomeric Heterochromatin inPlasmodium falciparum

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Rosaura Hernandez-Rivas ◽  
Karla Pérez-Toledo ◽  
Abril-Marcela Herrera Solorio ◽  
Dulce María Delgadillo ◽  
Miguel Vargas
Keyword(s):  
Plasmodium Falciparum ◽  
Drosophila Melanogaster ◽  
Gene Family ◽  
Histone Modifications ◽  
Chromatin Structure ◽  
Antigenic Variation ◽  
Histone H3 ◽  
Histone H4 ◽  
Telomeric Heterochromatin ◽  
Subtelomeric Regions

Until very recently, little was known about the chromatin structure of the telomeres and subtelomeric regions inPlasmodium falciparum. In yeast andDrosophila melanogaster, chromatin structure has long been known to be an important aspect in the regulation and functioning of these regions. Telomeres and subtelomeric regions are enriched in epigenetic marks that are specific to heterochromatin, such as methylation of lysine 9 of histone H3 and lysine 20 of histone H4. InP. falciparum, histone modifications and the presence of both the heterochromatin “writing” (PfSir2, PKMT) and “reading” (PfHP1) machinery at telomeric and subtelomeric regions indicate that these regions are likely to have heterochromatic structure that is epigenetically regulated. This structure may be important for telomere functions such as the silencing of thevargene family implicated in the cytoadherence and antigenic variation of these parasites.

scholarly journals Same, Same, but Different: Molecular Analyses of Streptococcus pneumoniae Immune Evasion Proteins Identifies new Domains and Reveals Structural Differences between PspC and Hic Variants

2020 ◽  
Author(s):  
Shanshan Du ◽  
Claudia Vilhena ◽  
Samantha King ◽  
Alfredo Sahagun ◽  
Sven Hammerschmidt ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Immune Evasion ◽  
Antigenic Variation ◽  
Immune Escape ◽  
Functional Characterization ◽  
Surface Proteins ◽  
Surface Distribution ◽  
Domain Composition ◽  
Structural Differences ◽  
Domain Level

AbstractPspC and Hic proteins of Streptococcus pneumoniae are some of the most variable microbial immune evasion proteins identified to date. Due to structural similarities and conserved binding profiles it was assumed over a long time that these pneumococcal surface proteins represent a protein family, comprising eleven subgroups. Recently, however, by evaluating more proteins larger diversity of individual proteins became apparent. In contrast to previous assumptions a pattern evaluation of six PspC and five Hic variants, each representing one of the previously defined subgroups, revealed distinct structural and likely functionally regions of the proteins, and identified nine new domains and new domain alternates. Several domains are unique to PspC and Hic variants, while other domains are shared with other S. pneumoniae and bacterial virulent determinants. This understanding improved pattern evaluation on the level of full-length proteins, allowed a sequence comparison on the domain level and furthermore identified domains with a modular composition. This novel concept allows a better characterization of variability, and modular domain composition of individual proteins, enables a structural and functional characterization at the domain level and furthermore shows substantial structural differences between PspC and Hic proteins. Such knowledge will also be useful for molecular strain typing, characterizing PspC and Hic proteins from new clinical S. pneumoniae strains, including those derived from patients who present with pneumococcal hemolytic uremic syndrome. Furthermore this analysis explains the role of multifaceted intact PspC and Hic proteins in pathogen host interactions. and can provide a basis for rational vaccine design.Author SummaryThe human pathobiont Streptococcus pneumoniae expresses highly polymorphic PspC or Hic proteins, which bind a repertoire of host immune regulators and combine antigenic variation with conserved immune evasion features. Understanding domain composition of each protein encoded by more than 60 000 pspC or hic genes deposited in the data banks defines their diversity, a role in immune escape and can furthermore delineate structure function approach for single protein domains. PspC and Hic proteins show variable domain composition and sequence diversity, which explain differences in binding of human regulators and likely in immune escape. The results of our analyses provide insights in the domain composition of these diverse immune evasion proteins, identifies new domains, defines domains which are unique to PspC or Hic variants, and identifies domains which are shared with other bacterial immune evasion proteins. These data have implication on cell wall attachment, surface distribution and in immune escape.

scholarly journals Nuclear Pores and Perinuclear Expression Sites of var and Ribosomal DNA Genes Correspond to Physically Distinct Regions in Plasmodium falciparum

2013 ◽  
Vol 12 (5) ◽  
pp. 697-702 ◽  
Author(s):  
Julien Guizetti ◽  
Rafael Miyazawa Martins ◽  
Stéphanie Guadagnini ◽  
Aurélie Claes ◽  
Artur Scherf
Keyword(s):  
Gene Expression ◽  
Plasmodium Falciparum ◽  
Gene Family ◽  
Ribosomal Dna ◽  
Monoallelic Expression ◽  
Nuclear Pore ◽  
Nuclear Surface ◽  
Nuclear Pores ◽  
Content Type ◽  
Expression Site

ABSTRACT The human malaria parasite Plasmodium falciparum modifies the erythrocyte it infects by exporting variant proteins to the host cell surface. The var gene family that codes for a large, variant adhesive surface protein called P. falciparum erythrocyte membrane protein 1 (PfEMP1) plays a particular role in this process, which is linked to pathogenesis and immune evasion. A single member of this gene family is highly transcribed while the other 59 members remain silenced. Importantly, var gene transcription occurs at a spatially restricted, but yet undefined, perinuclear site that is distinct from repressed var gene clusters. To advance our understanding of monoallelic expression, we investigated whether nuclear pores associate with the var gene expression site. To this end, we studied the nuclear pore organization during the asexual blood stage using a specific antibody directed against a subunit of the nuclear pore, P. falciparum Nup116 (PfNup116). Ring and schizont stage parasites showed highly polarized nuclear pore foci, whereas in trophozoite stage nuclear pores redistributed over the entire nuclear surface. Colocalization studies of var transcripts and anti-PfNup116 antibodies showed clear dissociation between nuclear pores and the var gene expression site in ring stage. Similar results were obtained for another differentially transcribed perinuclear gene family, the ribosomal DNA units. Furthermore, we show that in the poised state, the var gene locus is not physically linked to nuclear pores. Our results indicate that P. falciparum does form compartments of high transcriptional activity at the nuclear periphery which are, unlike the case in yeast, devoid of nuclear pores.

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