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Biology subjects

Harris, C. T.

Publications and source records attributed to Harris, C. T..

4 recordsLinked to original sources

A non-canonical sensing pathway mediates Plasmodium adaptation to AA deficiency

Eukaryotes have canonical pathways for responding to amino acid (AA) availability. Under AA-limiting conditions, the TOR complex is repressed, whereas the sensor kinase GCN2 is activated. While these pathways have been highly conserved throughout evolution, malaria parasites are a rare exception. Despite auxotrophic for most AA, Plasmodium does not have either a TOR complex nor the GCN2-downstream transcription factors. While Ile starvation has been shown to trigger eIF2 phosphorylation and a hibernatory-like response, the overall mechanisms mediating detection and response to AA fluctuation in the absence of such pathways has remained elusive. Here we show that Plasmodium parasites rely on an efficient sensing pathway to respond to AA fluctuations. A phenotypic screen of kinase knockout mutant parasites identified nek4, eIK1 and eIK2 - the last two clustering with the eukaryotic eIF2 kinases - as critical for Plasmodium to sense and respond to distinct AA-limiting conditions. Such AA-sensing pathway is temporally regulated by these kinases at distinct life cycle stages and allows parasites to actively fine-tune replication and development in response to AA availability. Collectively, our data identify a previously unknown set of heterogeneous responses to AA depletion, mediated by a complex mechanism that is critical for modulating parasite cell cycle and survival.

microbiology↗

Global Profiling of Histone Modifications in Plasmodium falciparum using CUT&RUN.

We recently adapted a CUT&RUN protocol for genome-wide profiling of chromatin modifications in the human malaria parasite Plasmodium. Using the step-by-step protocol described below, we were able to generate high quality profiles of multiple histone modifications using only a small fraction of the cells required for ChIPseq. Using antibodies against two commonly profiled histone modifications, H3K4me3 and H3K9me3, we show here that CUT&RUN profiling is highly reproducible and closely recapitulates previously published ChIPseq-based abundance profiles of histone marks. Finally, we show that CUT&RUN requires substantially lower sequencing coverage for accurate profiling compared to ChIPseq.

microbiology↗

Metabolic competition between lipid metabolism and histone methylation regulates sexual differentiation in human malaria parasites.

For Plasmodium falciparum, the most widespread and virulent malaria parasite that infects humans, persistence depends on continuous asexual replication in red blood cells, while transmission to their mosquito vector requires asexual blood-stage parasites to differentiate into non-replicating gametocytes. This decision is controlled by stochastic de-repression of a heterochromatin-silenced locus encoding PfAP2-G, the master transcription factor of sexual differentiation. The frequency of pfap2-g de-repression was shown to be responsive to extracellular phospholipid precursors but the mechanism linking these metabolites to epigenetic regulation of pfap2-g was unknown. Here we show that this response is mediated by metabolic competition for the methyl donor S-adenosylmethionine between histone methyltransferases and phosphoethanolamine methyltransferase, a critical enzyme in the parasites pathway for de novo phosphatidylcholine synthesis. When phosphatidylcholine precursors are scarce, increased consumption of SAM for de novo phosphatidylcholine synthesis impairs maintenance of the histone methylation responsible for silencing pfap2-g, increasing the frequency of derepression and sexual differentiation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/476397v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@5e2c69org.highwire.dtl.DTLVardef@b5a7d6org.highwire.dtl.DTLVardef@1e118aorg.highwire.dtl.DTLVardef@1191a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

Generation of Transgenic Human Malaria Parasites With Strong Fluorescence in the Transmission Stages

Malaria parasites have a complex life cycle that includes specialized stages for transmission between their mosquito and human hosts. These stages are an understudied part of the lifecycle yet targeting them is an essential component of the effort to shrink the malaria map. The human parasite Plasmodium falciparum is responsible for the majority of deaths due to malaria. Our goal was to generate transgenic P. falciparum lines that could complete the lifecycle and produce fluorescent transmission stages for more in-depth and high-throughput studies. Using zinc-finger nuclease technology to engineer a marker-free integration site, we generated three transgenic P. falciparum lines in which tdtomato or gfp were stably integrated into the genome. Expression was driven by either stage-specific peg4 and csp promoters or the constitutive ef1a promoter. Phenotypic characterization of these lines demonstrates that they complete the life cycle with high infection rates and give rise to fluorescent mosquito stages. The transmission stages are sufficiently bright for intra-vital imaging, flow cytometry and scalable screening of chemical inhibitors and potentially inhibitory antibodies.

microbiology↗