Mapping and functional analysis of heterochromatin protein 1 phosphorylation in the malaria parasite Plasmodium falciparum

Previous studies in model eukaryotes have demonstrated that phosphorylation of heterochromatin protein 1 (HP1) is important for dynamically regulating its various functions. However, in the malaria parasite Plasmodium falciparum both the function of HP1 phosphorylation and the identity of the protein kinases targeting HP1 are still elusive. In order to functionally analyze phosphorylation of P. falciparum HP1 (PfHP1), we first mapped PfHP1 phosphorylation sites by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of native PfHP1, which identified motifs from which potential kinases could be predicted; in particular, several phosphorylated residues were embedded in motifs rich in acidic residues, reminiscent of targets for P. falciparum casein kinase 2 (PfCK2). Secondly, we tested recombinant PfCK2 and a number of additional protein kinases for their ability to phosphorylate PfHP1 in in vitro kinase assays. These experiments validated our prediction that PfHP1 acts as a substrate for PfCK2. Furthermore, LC-MS/MS analysis showed that PfCK2 phosphorylates three clustered serine residues in an acidic motif within the central hinge region of PfHP1. To study the role of PfHP1 phosphorylation in live parasites we used CRISPR/Cas9-mediated genome editing to generate a number of conditional PfHP1 phosphomutants based on the DiCre/LoxP system. Our studies revealed that neither PfCK2-dependent phosphorylation of PfHP1, nor phosphorylation of the hinge domain in general, affect PfHP1's ability to localize to heterochromatin, and that PfHP1 phosphorylation in this region is dispensable for the proliferation of P. falciparum blood stage parasites

From contigs towards chromosomes: automatic improvement of long read assemblies (ILRA)

Recent advances in long read technologies not only enable large consortia to aim to sequence all eukaryotes on Earth, but they also allow individual laboratories to sequence their species of interest with relatively low investment. Long read technologies embody the promise of overcoming scaffolding problems associated with repeats and low complexity sequences, but the number of contigs often far exceeds the number of chromosomes and they may contain many insertion and deletion errors around homopolymer tracts. To overcome these issues, we have implemented the ILRA pipeline to correct long read-based assemblies. Contigs are first reordered, renamed, merged, circularized, or filtered if erroneous or contaminated. Illumina short reads are used subsequently to correct homopolymer errors. We successfully tested our approach by improving the genome sequences of Homo sapiens, Trypanosoma brucei, and Leptosphaeria spp., and by generating four novel Plasmodium falciparum assemblies from field samples. We found that correcting homopolymer tracts reduced the number of genes incorrectly annotated as pseudogenes, but an iterative approach seems to be required to correct more sequencing errors. In summary, we describe and benchmark the performance of our new tool, which improved the quality of novel long read assemblies up to 1 Gbp. The pipeline is available at GitHub: https://github.com/ThomasDOtto/ILRA

Plasmodium gametocytes display homing and vascular transmigration in the host bone marrow

Transmission of Plasmodium parasites to the mosquito requires the formation and development of gametocytes. Studies in infected humans have shown that only the most mature forms of Plasmodium falciparum gametocytes are present in circulation, whereas immature forms accumulate in the hematopoietic environment of the bone marrow. We used the rodent model Plasmodium berghei to study gametocyte behavior through time under physiological conditions. Intravital microscopy demonstrated preferential homing of early gametocyte forms across the intact vascular barrier of the bone marrow and the spleen early during infection and subsequent development in the extravascular environment. During the acute phase of infection, we observed vascular leakage resulting in further parasite accumulation in this environment. Mature gametocytes showed high deformability and were found entering and exiting the intact vascular barrier. We suggest that extravascular gametocyte localization and mobility are essential for gametocytogenesis and transmission of Plasmodium to the mosquito

A var gene upstream element controls protein synthesis at the level of translation initiation in Plasmodium falciparum

Clonally variant protein expression in the malaria parasite Plasmodium falciparum generates phenotypic variability and allows isogenic populations to adapt to environmental changes encountered during blood stage infection. The underlying regulatory mechanisms are best studied for the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 is encoded by the multicopy var gene family and only a single variant is expressed in individual parasites, a concept known as mutual exclusion or singular gene choice. var gene activation occurs in situ and is achieved through the escape of one locus from epigenetic silencing. Singular gene choice is controlled at the level of transcription initiation and var 5' upstream (ups) sequences harbour regulatory information essential for mutually exclusive transcription as well as for the trans-generational inheritance of the var activity profile. An additional level of control has recently been identified for the var2csa gene, where an mRNA element in the 5' untranslated region (5' UTR) is involved in the reversible inhibition of translation of var2csa transcripts. Here, we extend the knowledge on post-transcriptional var gene regulation to the common upsC type. We identified a 5' UTR sequence that inhibits translation of upsC-derived mRNAs. Importantly, this 5' UTR element efficiently inhibits translation even in the context of a heterologous upstream region. Further, we found var 5' UTRs to be significantly enriched in uAUGs which are known to impair the efficiency of protein translation in other eukaryotes. Our findings suggest that regulation at the post-transcriptional level is a common feature in the control of PfEMP1 expression in P. falciparum

An assay to probe <i>Plasmodium falciparum</i> growth, transmission stage formation and early gametocyte development

Conversion from asexual proliferation to sexual differentiation initiates the production of the gametocyte, which is the malaria parasite stage required for human-to-mosquito transmission. This protocol describes an assay designed to probe the effect of drugs or other perturbations on asexual replication, sexual conversion and early gametocyte development in the major human malaria parasite Plasmodium falciparum. Synchronized asexually replicating parasites are induced for gametocyte production by the addition of conditioned medium, and they are then exposed to the treatment of interest during sexual commitment or at any subsequent stage of early gametocyte development. Flow cytometry is used to measure asexual proliferation and gametocyte production via DNA dye staining and the gametocyte-specific expression of a fluorescent protein, respectively. This screening approach may be used to identify and evaluate potential transmission-blocking compounds and to further investigate the mechanism of sexual conversion in malaria parasites. The full protocol can be completed in 11 d

Investigation of heterochromatin protein 1 function in the malaria parasite Plasmodium falciparum using a conditional domain deletion and swapping approach

The human malaria parasite; Plasmodium falciparum; encodes a single ortholog of heterochromatin protein 1 (PfHP1) that plays a crucial role in the epigenetic regulation of various survival-related processes. PfHP1 is essential for parasite proliferation and the heritable silencing of genes linked to antigenic variation, host cell invasion, and sexual conversion. Here, we employed CRISPR/Cas9-mediated genome editing combined with the DiCre/loxP system to investigate how the PfHP1 chromodomain (CD), hinge domain, and chromoshadow domain (CSD) contribute to overall PfHP1 function. We show that the 76 C-terminal residues are responsible for targeting PfHP1 to the nucleus. Furthermore, we reveal that each of the three functional domains of PfHP1 are required for heterochromatin formation, gene silencing, and mitotic parasite proliferation. Finally, we discovered that the hinge domain and CSD of HP1 are functionally conserved between; P. falciparum; and; P. berghei; , a related malaria parasite infecting rodents. In summary, our study provides new insights into PfHP1 function and offers a tool for further studies on epigenetic regulation and life cycle decision in malaria parasites.; IMPORTANCE; Malaria is caused by unicellular; Plasmodium; species parasites that repeatedly invade and replicate inside red blood cells. Some blood-stage parasites exit the cell cycle and differentiate into gametocytes that are essential for malaria transmission via the mosquito vector. Epigenetic control mechanisms allow the parasites to alter the expression of surface antigens and to balance the switch between parasite multiplication and gametocyte production. These processes are crucial to establish chronic infection and optimize parasite transmission. Here, we performed a mutational analysis of heterochromatin protein 1 (HP1) in; P. falciparum; We demonstrate that all three domains of this protein are indispensable for the proper function of HP1 in parasite multiplication, heterochromatin formation, and gene silencing. Moreover, expression of chimeric proteins revealed the functional conservation of HP1 proteins between different; Plasmodium; species. These results provide new insight into the function and evolution of HP1 as an essential epigenetic regulator of parasite survival

The MEE inhibits translation in the natural context of the <i>upsC</i> promoter.

<p>(A) Schematic depiction of <i>upsC var</i> promoter reporter constructs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100183#pone.0100183-Brancucci1" target="_blank">[54]</a>. Deletions are represented by dashed lines. Numbers refer to the nucleotide positions relative to the ATG start codon. The position of the MEE is highlighted. (B) Expression of hDHFR-GFP and GAPDH (loading control) in WR-selected parasites was analysed by semi-quantitative Western blot. (C) Top panel: Proportion of total steady-state h<i>dhfr-gfp</i> transcripts in WR-selected parasites carrying truncated upstream sequences relative to the control line 3D7/pBC. Values are derived from three independent experiments (mean +/− s.d.) (normalised to PF3D7_1331700 transcripts). Middle panel: Proportion of steady-state h<i>dhfr-gfp</i> transcripts produced by a single promoter in WR-selected parasites carrying truncated upstream sequences relative to the control line 3D7/pBC. Values represent the data displayed in the top panel divided by the average plasmid copy number determined from the same batch of parasites (bottom panel). (D) Mean increase in plasmid copy numbers (+/− s.d.) after WR selection in parasites transfected with constructs carrying MEE-positive upstream sequences (red) or MEE-negative upstream sequences (green). The increase in plasmid copy numbers in WR-selected 3D7/pBC4 is shown in black. Individual plasmid copy numbers determined for each population are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100183#pone.0100183.s003" target="_blank">Figure S3</a>. Asterisk, p = 0.0015 (Student’s t-test).</p

Integration of the <i>upsC</i> 5′ upstream sequence into a heterologous context at the <i>kahrp</i> locus.

<p>(A) Schematic map of the transfection construct pBK_minC. Single-crossover integration was guided by <i>kahrp</i> 5′ homology. The position of the <i>kahrp</i> TSS is indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100183#pone.0100183-Lanzer1" target="_blank">[81]</a>. Numbers refer to the nucleotide positions relative to the ATG start codon. The <i>bsd</i> resistance cassette selects for stably transfected parasites. The <i>var</i> intron is indicated by a bold dashed line. hsp86 5′, <i>hsp86</i> promoter; Pb DT 3′, <i>P. berghei dhfr</i>-thymidylate synthase terminator; rep20, 0.5 kb TARE6 repeat element; hrp2 3′; histidine-rich protein 2 terminator. MEE, location of the 101 bp mutual exclusion element MEE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100183#pone.0100183-Brancucci1" target="_blank">[54]</a>. (B) Genomic situation after integration of the pBK_minC concatamer into the endogenous <i>kahrp</i> locus. Restriction sites used in Southern analysis and fragment lengths are indicated and colour-coded. S, <i>Stu</i>I; B, <i>Bgl</i>II. The Southern blot on <i>Bgl</i>II/<i>Stu</i>I-digested gDNA shows integration of pBK_minC into the endogenous locus of <i>kahrp</i>. The membrane was hybridised with h<i>dhfr</i> (top) and <i>kahrp</i> (bottom). Fragments are colour-coded according to the integration map. wt, size of the <i>kahrp</i> fragment in 3D7 wild-type parasites. i, integration event; p, plasmid fragment. (C) The <i>upsC</i> 5′ UTR sequence represses <i>kahrp</i> promoter activity. The bars represent the ratio of relative h<i>dhfr-gfp</i> and <i>msp8</i> transcript levels in 3D7/pBK_minC parasites (open bars) compared to the 3D7/pBK_min control (black bars) cultured in absence of WR. Results are the mean +/− s.d. of three independent experiments. Values are normalised for PF3D7_1331700 transcripts.</p

The <i>upsC</i> 5′ UTR element inhibits translation.

<p>Semi-quantitative analysis of transcript and protein abundance in 3D7/pBK_min (control) and 3D7/pBK_minC ring stage parasites (6–14 hpi) cultured in absence of WR (−WR). Top panels: h<i>dhfr-gfp</i> and <i>hsp86</i> (loading control) transcripts were detected by Northern blot. Ethidium bromide-stained 18S and 28S rRNAs serve as second loading control. Bottom panels: expression of hDHFR-GFP and GAPDH (loading control) in the same parasite samples were analysed by Western blot.</p

A gene conversion event revokes translational inhibition of h<i>dhfr-gfp</i> transcripts.

<p>(A) Southern analysis on digested gDNA from unselected and WR-selected 3D7/pBK_minC parasites. Additional h<i>dhfr</i>-containing fragments detected in WR-selected parasites only are highlighted by pink arrows. S, <i>Stu</i>I; B, <i>Bgl</i>II; K, <i>Kpn</i>I; i, integration event; p, plasmid fragment. (B) The ends of chromosome 2 and 4 in unselected and 4/2 in WR-selected parasites are schematically depicted. Gene IDs (<a href="http://www.plasmoDB.org" target="_blank">www.plasmoDB.org</a>) are indicated for a subset of genes as reference. The dashed arrow highlights the site of gene conversion. The blue box represents the duplicated region of chromosome 2. The green box represents the region of chromosome 4 that was deleted. The brown box displays a zoom-in view of the gene conversion event and the resulting recombined locus. Detailed mapping and identification of the recombination site is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100183#pone.0100183.s001" target="_blank">Figures S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100183#pone.0100183.s002" target="_blank">S2</a>. (C) h<i>dhfr-gfp</i> transcripts are produced from the <i>var</i> gene intron on chromosome 4 in WR-selected 3D7/pBK_minC parasites. Values represent relative <i>var</i> intron-derived h<i>dhfr-gfp</i> (grey bars) and ring stage-specific <i>msp8</i> (open bars, control) transcript levels at three consecutive time points in WR-selected 3D7/pBK_minC parasites (normalised to PF3D7_1331700 transcripts). hpi, hours post invasion. (D) Semi-quantitative analysis of transcript and protein abundance in 3D7/pBK_min (control) and 3D7/pBK_minC ring stage parasites (6–14 hpi) cultured in presence of WR99210 (+WR). Top panels: h<i>dhfr-gfp</i> and <i>hsp86</i> (loading control) transcripts were detected by Northern blot. Ethidium bromide-stained 18S and 28S rRNAs serve as second loading control. Bottom panels: expression of hDHFR-GFP and GAPDH (loading control) in the same parasite samples were analysed by Western blot.</p