C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites

Anopheles gambiae melanization-based refractoriness to the human malaria parasite Plasmodium falciparum has rarely been observed in either laboratory or natural conditions, in contrast to the rodent model malaria parasite Plasmodium berghei that can become completely melanized by a TEP1 complement-like system-dependent mechanism. Multiple studies have shown that the rodent parasite evades this defense by recruiting the C-type lectins CTL4 and CTLMA2, while permissiveness to the human malaria parasite was not affected by partial depletion of these factors by RNAi silencing. Using CRISPR/Cas9-based CTL4 knockout, we show that A. gambiae can mount melanization-based refractoriness to the human malaria parasite, which is independent of the TEP1 complement-like system and the major anti-Plasmodium immune pathway Imd. Our study indicates a hierarchical specificity in the control of Plasmodium melanization and proves CTL4 as an essential host factor for P. falciparum transmission and one of the most potent mosquito-encoded malaria transmission-blocking targets.

Variable oxygen environments and DNMT2 determine the DNA cytosine epigenetic landscape of Plasmodium falciparum

DNA cytosine methylation and its oxidized products are important epigenetic modifications in mammalian cells. Although 5-methylcytosine (5mC) was detected in the human malaria parasite Plasmodium falciparum, the presence of oxidized 5mC forms remain to be characterized.Here we establish a protocol to explore nuclease-based DNA digestion for the extremely AT-rich genome of P. falciparum (>80% A+T) for quantitative LC-MS/MS analysis of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). We demonstrate the presence of 5hmC, 5fC and 5caC cytosine modifications in a DNMT2-only organism and observe striking ratio changes between 5mC and 5hmC during the 48-hour blood stage parasite development. Parasite-infected red blood cells cultured in different physiological oxygen concentrations revealed a shift in the cytosine modifications distribution towards the oxidized 5hmC and 5caC forms. In the absence of the canonical C5-DNA methyltransferase (DNMT1 and DNMT3A/B) in P. falciparum, we show that all cytosine modifications depend on the presence of DNMT2. We conclude that DNMT2 and oxygen levels are critical determinants that shape the dynamic cytosine epigenetic landscape in this human pathogen.

Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of “pseudoschizonts,” which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

Localization of the putative recombinase Pf-int to the apicoplast of Plasmodium falciparum

Diseases caused by apicomplexan parasites, such as malaria and toxoplasmosis cause ∼200 million (worldwide) and 1 million (Europe) infections, respectively, every year. Apicomplexa possess a non-photosynthetic organelle homologous to the plant chloroplast, the so-called apicoplast, that is essential for their growth and survival. This study focused on the Int recombinase, the first protein discovered in Plasmodium spp. with the features of a site-specific recombinase, and which has an apicoplast targeting leader sequence at its amino-terminus. Int is conserved amongst several apicomplexan parasites. In the human toxoplasmosis parasite, Toxoplasma, Int localizes to the apicoplast and Pf-Int, the P. falciparum member, belongs to the group of non-mutable essential genes in P. falciparum. A conserved protein that has been shown to be essential at least in one species and that localizes to an essential organelle may become a novel drug target. Therefore, the aim of this study was to confirm the sub-cellular localization of Int in the human malaria parasite P. falciparum. Using western blot analysis and immunofluorescence microscopy of P. falciparum asexual blood stages, we observed that Int partially co-localized with the apicoplast (to discrete foci adjacent to the nucleus).

A New Thienopyrimidinone Chemotype Shows Multistage Activity against Plasmodium falciparum, Including Artemisinin-Resistant Parasites

This work reports a new chemical structure that (i) displays activity against the human malaria parasite Plasmodium falciparum at 3 stages of the parasitic cycle (blood stage, hepatic stage, and sexual stages), (ii) remains active against parasites that are resistant to the first-line treatment recommended by the World Health Organization (WHO) for the treatment of severe malaria (artemisinins), and (iii) reduces transmission of the parasite to the mosquito vector in a mouse model. This new molecule family could open the way to the conception of novel antimalarial drugs with an original multistage mechanism of action to fight against Plasmodium drug resistance and block interhuman transmission of malaria.

Real-time PCR assays for detection and quantification of early P. falciparum gametocyte stages

The use of quantitative qRT-PCR assays for detection and quantification of late gametocyte stages has revealed the high transmission capacity of the human malaria parasite, Plasmodium falciparum. To understand how the parasite adjusts its transmission in response to in-host environmental conditions including antimalarials requires simultaneous quantification of early and late gametocytes. Here, we describe qRT-PCR assays that specifically detect and quantify early-stage P. falciparum gametocytes. The assays are based on expression of known early and late gametocyte genes and were developed using purified stage II and stage V gametocytes and tested in natural and controlled human infections. Genes pfpeg4 and pfg27 are specifically expressed at significant levels in early gametocytes with a limit of quantification of 190 and 390 gametocytes/mL, respectively. In infected volunteers, transcripts of pfpeg4 and pfg27 were detected shortly after the onset of blood stage infection. In natural infections, both early (pfpeg4/pfg27) and late gametocyte transcripts (pfs25) were detected in 71.2% of individuals, only early gametocyte transcripts in 12.6%, and only late gametocyte transcripts in 15.2%. The pfpeg4/pfg27 qRT-PCR assays are sensitive and specific for quantification of circulating sexually committed ring stages/early gametocytes and can be used to increase our understanding of epidemiological processes that modulate P. falciparum transmission.

Identifying transcript 5′ capped ends in Plasmodium falciparum

Background The genome of the human malaria parasite Plasmodium falciparum is poorly annotated, in particular, the 5′ capped ends of its mRNA transcripts. New approaches are needed to fully catalog P. falciparum transcripts for understanding gene function and regulation in this organism. Methods We developed a transcriptomic method based on next-generation sequencing of complementary DNA (cDNA) enriched for full-length fragments using eIF4E, a 5′ cap-binding protein, and an unenriched control. DNA sequencing adapter was added after enrichment of full-length cDNA using two different ligation protocols. From the mapped sequence reads, enrichment scores were calculated for all transcribed nucleotides and used to calculate P-values of 5′ capped nucleotide enrichment. Sensitivity and accuracy were increased by combining P-values from replicate experiments. Data were obtained for P. falciparum ring, trophozoite and schizont stages of intra-erythrocytic development. Results 5′ capped nucleotide signals were mapped to 17,961 non-overlapping P. falciparum genomic intervals. Analysis of the dominant 5′ capped nucleotide in these genomic intervals revealed the presence of two groups with distinctive epigenetic features and sequence patterns. A total of 4,512 transcripts were annotated as 5′ capped based on the correspondence of 5′ end with 5′ capped nucleotide annotated from full-length cDNA data. Discussion The presence of two groups of 5′ capped nucleotides suggests that alternative mechanisms may exist for producing 5′ capped transcript ends in P. falciparum. The 5′ capped transcripts that are antisense, outside of, or partially overlapping coding regions may be important regulators of gene function in P. falciparum.

4D live-cell imaging of microgametogenesis in the human malaria parasite Plasmodium falciparum

Formation of gametes in the malaria parasite occurs in the midgut of the mosquito and is critical to onward parasite transmission. Transformation of the male gametocyte into microgametes, called microgametogenesis, is an explosive cellular event and one of the fastest eukaryotic DNA replication events known. The transformation of one microgametocyte into eight flagellated microgametes requires reorganisation of the parasite cytoskeleton, replication of the 22.9 Mb genome, axoneme formation and host erythrocyte egress, all of which occur simultaneously in <20 minutes. Whilst high-resolution imaging has been a powerful tool for defining stages of microgametogenesis, it has largely been limited to fixed parasite samples, given the speed of the process and parasite photosensitivity. Here, we have developed a live-cell fluorescence imaging workflow that captures the explosive dynamics of microgametogenesis in full. Using the most virulent human malaria parasite, Plasmodium falciparum, our live-cell approach combines three-dimensional imaging through time (4D imaging) and covers early microgametocyte development through to microgamete release. Combining live-cell stains for DNA, tubulin and the host erythrocyte membrane, 4D imaging enables definition of the positioning of newly replicated and segregated DNA. It also shows the microtubular cytoskeleton, location of newly formed basal bodies and elongation of axonemes, as well as behaviour of the erythrocyte membrane, including its specific perforation prior to microgamete egress. 4D imaging was additionally undertaken in the presence of known transmission-blocking inhibitors and the untested proteasomal inhibitor bortezomib. Here, for the first time we find that bortezomib inhibition results in a clear block of DNA replication, full axoneme nucleation and elongation. These data not only define a framework for understanding microgametogenesis in general but also suggest that the process is critically dependent on proteasomal activity, helping to identify potentially novel targets for transmission-blocking antimalarial drug development.