Heterochromatin formation in bistable chromatin domains controls the epigenetic repression of clonally variant Plasmodium falciparum genes linked to erythrocyte invasion

Clonally variant gene expression is a common survival strategy used by many pathogens, including the malaria parasite Plasmodium falciparum. Among the genes that show variant expression in this parasite are several members of small gene families linked to erythrocyte invasion, including the clag and eba families. The active or repressed state of these genes is clonally transmitted by epigenetic mechanisms. Here we characterized the promoters of clag3.1, clag3.2 and eba-140, and compared nuclease accessibility and post-translational histone modifications between their active and repressed states. Activity of these promoters in an episomal context is similar between parasite subclones characterized by different patterns of expression of the endogenous genes. Variant expression is controlled by the euchromatic or heterochromatic state of bistable chromatin domains. Repression is mediated by H3K9me3-based heterochromatin, whereas the active state is characterized by H3K9ac. These marks are maintained throughout the asexual blood cycle to transmit the epigenetic memory. Furthermore, eba-140 is organized in two distinct chromatin domains, probably separated by a barrier insulator located within its ORF. The 5' chromatin domain controls expression of the gene, whereas the 3' domain shares the chromatin conformation with the upstream region of the neighbouring phista family gene, which also shows variant expression.     

5' flanking region of var genes nucleate histone modification patterns linked to phenotypic inheritance of virulence traits in malaria parasites

In the human malaria parasite Plasmodium falciparum antigenic variation facilitates long-term chronic infection of the host. This is achieved by sequential expression of a single member of the 60-member var family. Here we show that the 5' flanking region nucleates epigenetic events strongly linked to the maintenance of mono-allelic var gene expression pattern during parasite proliferation. Tri- and dimethylation of histone H3 lysine 4 peak in the 5' upstream region of transcribed var and during the poised state (non-transcribed phase of var genes during the 48 h asexual life cycle), 'bookmarking' this member for re-activation at the onset of the next cycle. Histone H3 lysine 9 trimethylation acts as an antagonist to lysine 4 methylation to establish stably silent var gene states along the 5' flanking and coding region. Furthermore, we show that competition exists between H3K9 methylation and H3K9 acetylation in the 5' flanking region and that these marks contribute epigenetically to repressing or activating var gene expression. Our work points to a pivotal role of the histone methyl mark writing and reading machinery in the phenotypic inheritance of virulence traits in the malaria parasite.     

Effect of heat-shock on Plasmodium falciparum viability, growth and expression of the heat-shock protein 'PFHSP70-I' gene.

Cultures of the human malaria parasite Plasmodium falciparum were subjected to heat-shock for varying times and temperatures and then tested for their viability, growth and expression of heat-shock protein. Results show that the majority of parasites remained viable after heat-shock but their growth was affected. However, the expression of the heat-shock protein 'PFHSP70-I' gene was enhanced after heat-shock. We conclude that malarial parasites are able to survive in vivo during fever probably due to the overexpression of the heat-shock protein gene.     

New technologies to study helminth development and host-parasite interactions

How parasites develop and survive, and how they stimulate or modulate host immune responses are important in understanding disease pathology and for the design of new control strategies. Microarray analysis and bulk RNA sequencing have provided a wealth of data on gene expression as parasites develop through different life-cycle stages and on host cell responses to infection. These techniques have enabled gene expression in the whole organism or host tissue to be detailed, but do not take account of the heterogeneity between cells of different types or developmental stages, nor the spatial organisation of these cells. Single-cell RNA-seq (scRNA-seq) adds a new dimension to studying parasite biology and host immunity by enabling gene profiling at the individual cell level. Here we review the application of scRNA-seq to establish gene expression cell atlases for multicellular helminths and to explore the expansion and molecular profile of individual host cell types involved in parasite immunity and tissue repair. Studying host-parasite interactions in vivo is challenging and we conclude this review by briefly discussing the applications of organoids (stem-cell derived mini-tissues) to examine host-parasite interactions at the local level, and as a potential system to study parasite development in vitro. Organoid technology and its applications have developed rapidly, and the elegant studies performed to date support the use of organoids as an alternative in vitro system for research on helminth parasites.     

Plasmodium falciparum variant surface antigen expression patterns during malaria

The variant surface antigens expressed on Plasmodium falciparum-infected erythrocytes are potentially important targets of immunity to malaria and are encoded, at least in part, by a family of var genes, about 60 of which are present within every parasite genome. Here we use semi-conserved regions within short var gene sequence "tags" to make direct comparisons of var gene expression in 12 clinical parasite isolates from Kenyan children. A total of 1,746 var clones were sequenced from genomic and cDNA and assigned to one of six sequence groups using specific sequence features. The results show the following. (1) The relative numbers of genomic clones falling in each of the sequence groups was similar between parasite isolates and corresponded well with the numbers of genes found in the genome of a single, fully sequenced parasite isolate. In contrast, the relative numbers of cDNA clones falling in each group varied considerably between isolates. (2) Expression of sequences belonging to a relatively conserved group was negatively associated with the repertoire of variant surface antigen antibodies carried by the infected child at the time of disease, whereas expression of sequences belonging to another group was associated with the parasite "rosetting" phenotype, a well established virulence determinant. Our results suggest that information on the state of the host-parasite relationship in vivo can be provided by measurements of the differential expression of different var groups, and need only be defined by short stretches of sequence data.     

Genomic data reveal Toxoplasma gondii differentiation mutants are also impaired with respect to switching into a novel extracellular tachyzoite state

Toxoplasma gondii pathogenesis includes the invasion of host cells by extracellular parasites, replication of intracellular tachyzoites, and differentiation to a latent bradyzoite stage. We present the analysis of seven novel T. gondii insertional mutants that do not undergo normal differentiation to bradyzoites. Microarray quantification of the variation in genome-wide RNA levels for each parasite line and times after induction allowed us to describe states in the normal differentiation process, to analyze mutant lines in the context of these states, and to identify genes that may have roles in initiating the transition from tachyzoite to bradyzoite. Gene expression patterns in wild-type parasites undergoing differentiation suggest a novel extracellular state within the tachyzoite stage. All mutant lines exhibit aberrant regulation of bradyzoite gene expression and notably some of the mutant lines appear to exhibit high proportions of the intracellular tachyzoite state regardless of whether they are intracellular or extracellular. In addition to the genes identified by the insertional mutagenesis screen, mixture model analysis allowed us to identify a small number of genes, in mutants, for which expression patterns could not be accounted for using the three parasite states--genes that may play a mechanistic role in switching from the tachyzoite to bradyzoite stage.     

The H89 cAMP-dependent protein kinase inhibitor blocks Plasmodium falciparum development in infected erythrocytes.

In Plasmodium falciparum, the causative agent of human malaria, the catalytic subunit gene of cAMP-dependent protein kinase (Pfpka-c) exists as a single copy. Interestingly, its expression appears developmentally regulated, being at higher levels in the pathogenic asexual stages than in the sexual forms of parasite that are responsible for transmission to the mosquito vector. Within asexual parasites, PfPKA activity can be readily detected in schizonts. Similar to endogenous PKA activity of noninfected red blood cells, the parasite enzyme can be stimulated by cAMP and inhibited by protein kinase inhibitor.Importantly, ex vivo treatment of infected erythrocytes with the classical PKA-C inhibitor H89 leads to a block in parasite growth. This suggests that the PKA activities of infected red blood cells are essential for parasite multiplication. Finally, structural considerations suggest that drugs targeting the parasite, rather than the erythrocyte enzyme, might be developed that could help in the fight against malaria.     

Chromatin modifications, epigenetics, and how protozoan parasites regulate their lives

Chromatin structure plays a vital role in epigenetic regulation of protozoan parasite gene expression. Epigenetic gene regulation impacts upon parasite virulence, differentiation and cell-cycle control. Recent work in many laboratories has elucidated the functions of proteins that regulate parasite gene expression by chemical modification of constituent nucleosomes. A major focus of investigation has been the characterization of post-translational modifications (PTMs) of histones and the identification of the enzymes responsible. Despite conserved features and specificity common to all eukaryotes, parasite enzymes involved in chromatin modification have unique functions that regulate unique aspects of parasite biology.     

Development of hammerhead ribozymes to modulate endogenous gene expression for functional studies

Hammerhead ribozymes are catalytic RNAs that are being used to inhibit endogenous gene expression to study key components of basic biochemical pathways such as angiogenesis. In addition, these ribozymes have the potential to be used as components of gene therapy protocols for the treatment of disease states. We detail here a set of protocols for the design and testing of hammerhead ribozymes that will efficiently inhibit gene expression both in cell culture and in vivo.     

In vivo gene silencing in Plasmodium berghei--a mouse malaria model

RNA interference (RNAi) has emerged as a specific and efficient tool to silence gene expression in a variety of organisms and cell lines. An important prospect for RNAi technology is its possible application in the treatment of diseases using short interfering RNAs (siRNAs). However, the effect of siRNAs in adult animals and their potential to treat or prevent diseases are yet to be fully investigated. The main goal of the present study is to find out whether it was possible to carry out RNAi on circulating malaria parasite in vivo. To trigger RNAi in mouse malaria parasite, we used siRNAs corresponding to cysteine protease genes of Plasmodium berghei (berghepain-1 & 2). Intravenous injections of berghepains' siRNAs in infected animal resulted in characteristic enlargement of food vacuole in circulating parasites. Protein analysis of these treated parasites showed substantial accumulation of hemoglobin, which is reminiscent of the effect observed upon treating Plasmodium falciparum with different cysteine protease inhibitors. Parasites treated with berghepain 1 & 2 siRNAs showed marked reduction in the levels of their cognate mRNAs, thereby suggesting specific inhibition of berghepains' gene expression in vivo. We also observed the generation of approximately 25 nt RNA species from berghepains' mRNAs in the treated parasites, which is a characteristic of an RNAi phenomenon. These results thus provide evidence that beyond its value for validation of gene functions, RNAi may provide a new approach for disease therapy.     

Nicotinamide inhibits Plasmodium falciparum Sir2 activity in vitro and parasite growth

Plasmodium falciparum sirtuin, PfSir2, contains histone deacetylase (HDAC) activity that may be central to the regulation of virulence gene expression in the parasites. Although a few reports have been published recently regarding in vitro and in vivo function of PfSir2, expression of the endogenous protein (c. 30 kDa) has not been shown yet. Here we report the presence of PfSir2 in the parasite at the protein level by specific antibodies. HDAC activity of PfSir2 can be inhibited by nicotinamide, a product of sirtuin reaction. Surprisingly, we find that nicotinamide also delays parasite growth significantly in culture. These findings further our knowledge on PfSir2 and raise the possibility of using an inexpensive agent like nicotinamide as an antimalarial in combination with other antiparasitic drugs.     

A Bovine Lymphosarcoma Cell Line Infected with Theileria annulata Exhibits an Irreversible Reconfiguration of Host Cell Gene Expression

Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner.     

The genetic toolbox for Leishmania parasites

Leishmania parasites cause a variety of devastating diseases in tropical areas around the world. Due to the lack of vaccines and limited availability of drugs, new therapeutic targets are urgently needed. A variety of genetic tools have been developed to investigate the complex biology of this parasite and its interactions with the host. One of the main techniques is the generation of knock-out parasites via targeted gene replacement, a process that takes advantage of the parasites ability to undergo homologous recombination. Studying the effect of gene deletions in vitro and in infectivity models in vivo allows understanding the function of a target gene and its potential as a therapeutic target. Other genetic manipulations available include episomal and chromosomal complementation and the generation of overproducer strains. However, there are also limitations, such as the lack of RNA interference machinery in most Leishmania species and limited options for inducible expression systems. The genomes of several Leishmania species have now been sequenced and will provide powerful resources in combination with the genetic tools that are available. The increasing knowledge of parasite biology and host parasite interactions derived from these studies will raise the number of potential therapeutic targets, which are sorely needed to combat leishmaniasis.     

Theoretical design of a gene therapy to prevent AIDS but not human immunodeficiency virus type 1 infection

Recent reports confirm that, due to the presence of long-lived, latently infected cell populations, eradication of human immunodeficiency virus type 1 (HIV-1) from infected patients by using antiretroviral drugs will be exceedingly difficult. An alternative to virus eradication may be to use gene therapy to induce a pseudo-latent state in virus-producing cells, thus transforming HIV-1 into a lifelong, but manageable, virus. Conditionally replicating HIV-1 (crHIV-1) gene therapy vectors provide an avenue for subduing HIV-1 expression in infected cells (by creating a parasite, crHIV-1, of the parasite HIV-1), potentially reducing the HIV-1 set point and delaying AIDS onset. Development of crHIV-1 vectors has proceeded in vitro, but the requirements for a crHIV-1 vector to proliferate and persist in vivo have not been explored. We expand a widely accepted mathematical model of HIV-1 in vivo dynamics to include a crHIV-1 gene therapy virus and derive a simple criterion for designing crHIV-1 viruses that will persist in vivo. The model introduces only two new parameters-HIV-1 inhibition and crHIV-1 production-and both can be experimentally engineered and controlled. Analysis demonstrates that crHIV-1 gene therapy can indefinitely reduce HIV-1 set point to levels comparable to those achieved with highly active antiretroviral therapy, provided crHIV-1 production is more efficient than HIV-1. Paradoxically, highly efficient therapeutic inhibition of HIV-1 was found to be disadvantageous. Thus, the field may benefit by shifting the search for more potent antiviral genes toward engineering optimized therapy viruses that package ultra-efficiently while downregulating viral production moderately.     

Molecular diversity of the Trypanosoma cruzi TcSMUG family of mucin genes and proteins

The surface of the protozoan Trypanosoma cruzi is covered by a dense coat of mucin-type glycoconjugates, which make a pivotal contribution to parasite protection and host immune evasion. Their importance is further underscored by the presence of >1000 mucin-like genes in the parasite genome. In the present study we demonstrate that one such group of genes, termed TcSMUG L, codes for previously unrecognized mucin-type glycoconjugates anchored to and secreted from the surface of insect-dwelling epimastigotes. These features are supported by the in vivo tracing and characterization of endogenous TcSMUG L products and recombinant tagged molecules expressed by transfected parasites. Besides displaying substantial homology to TcSMUG S products, which provide the scaffold for the major Gp35/50 mucins also present in insect-dwelling stages of the T. cruzi lifecycle, TcSMUG L products display unique structural and functional features, including being completely refractory to sialylation by parasite trans-sialidases. Although quantitative real time-PCR and gene sequencing analyses indicate a high degree of genomic conservation across the T. cruzi species, TcSMUG L product expression and processing is quite variable among different parasite isolates.