Manuel Llinás

Understanding the molecular mechanisms of gene regulation and metabolism in the malaria parasite Plasmodium falciparum using functional genomics and metabolomics

Research Summary

Malaria is one of the most devastating diseases of humankind, affecting nearly one in ten people worldwide and resulting in over 1.5 million deaths annually. This disease is caused by the Plasmodium parasite, of which Plasmodium falciparum is the deadliest form. While the past century has seen significant progress in anti-malarial drug development, many of these drugs are losing efficacy due to the rise of drug-resistant parasites. One of the major challenges facing the field is the identification of new drug targets for efficacious, affordable treatment. My lab focuses on transcriptional regulation and metabolism as potential avenues to disrupt the progression of this deadly parasite. To accomplish this, our research combines tools from functional genomics, molecular biology, computational biology, biochemistry, and metabolomics to understand the fundamental molecular mechanisms underlying the development of this parasite. The focus is predominantly on the red blood cell stage of development, which is the stage in which all of the clinical manifestations of the malaria disease occur. Transcriptional regulation in malaria parasites: My lab is interested in role of transcriptional regulation in parasite development. To study this, we focus on the only known family of DNA binding proteins encoded by the Plasmodium genome, the Apicomplexan AP2 (ApiAP2) protein family. These proteins are highly conserved among all Apicomplexan parasites and find their origin in plants. (Why is there a plant connection you ask? Intriguingly, malaria parasites contain an amazing non-photosynthetic chloroplast-like organelle called the apicoplast which was acquired via a secondary endosymbiotic event so there are many features of plant cells in these intriguing single celled eukaryotic parasites!) To address the specific in vivo roles for the 27 members of the ApiAP2 protein family, we are pursuing several lines of inquiry. These approaches include modulating expression levels of these proteins, generating knockdowns and knockouts, as well as in vivo protein tagging for chromatin immunoprecipitation. We are also using luciferase reporter assays to measure the stage-specificity of expression controlled by the identified target DNA motifs, and we are using mass spectrometry-based proteomics to determine protein-protein interactions for these transcriptional regulatory complexes. Our goal is to define the dynamic transcriptional regulatory network of the malaria parasite and to determine which ApiAP2 proteins are the master regulators governing the various stages of parasite development including the blood stage, the mosquito stage and the liver stage with the goal of targeting these proteins as a way to kill parasites. Plasmodium metabolomics: The genome of Plasmodium falciparum indicates that the metabolic pathways utilized by this organism are highly unique. Recent efforts to comprehensively examine the biology of P. falciparum have focused on transcriptome and proteome analysis to gain insight into Plasmodium-specific pathways. The third crucial component that remains to be established is the metabolome: the complement of small-molecule metabolites and their relative levels. Our lab has begun to characterize various aspects of parasite metabolism using high accuracy mass-spectrometry to simultaneously measure metabolites from complex cellular extracts from parasite-infected cells. The approaches we are using allow us to assay various aspects of the P. falciparum metabolome. One approach has been to examine the interaction of Plasmodium with the host red blood cell using targeted measurements of specific metabolites shared with the host erythrocyte and asking how these vary when parasites are stressed or exposed to antimalarial drugs. We are also using 13C and 15N isotopic labeling experiments to directly trace carbon flux through known biochemical pathways. Finally, we are using metabolite measurements to map genetic control of metabolism by assaying global metabolite patterns in the parents and progeny of a Plasmodium falciparum genetic cross. Results from these studies are beginning to unravel the divergence of metabolism in P. falciparum and promise to provide unique avenues for future drug intervention strategies.

Selected Publications

Cowell AN, et al. “Mapping the malaria parasite druggable genome by using in vitro evolution and chemogenomics.” Science. 2018 January 12. doi: 10.1126/science.aan4472. Pubmed link to this paper.
Painter HJ, Chung NC, Sebastian A, Albert I, Storey JD, Llinás M. “Genome-wide real-time in vivo transcriptional dynamics during Plasmodium falciparum blood-stage development.” Nature Communications. 2018 Jul 9;9(1):2656. Pubmed link to this paper.
Santos JM, Josling G, Ross P, Joshi P, Orchard L, Campbell T, Schieler A, Cristea IM, Llinás M. “Red blood cell invasion by the malaria parasite is coordinated by the PfAP2-I transcription factor.” Cell Host and Microbe. 2017 Jun 14;21(6):731-741.e10. doi: 10.1016/j.chom.2017.05.006. Pubmed link to this paper.
Allman EL, Painter HJ, Samra J, Carrasquilla M, Llinás M. “Metabolomic Profiling of the Malaria Box Reveals Antimalarial Target Pathways.” Antimicrobial Agents and Chemotherapy. 2016 Aug 29; pii: AAC.01224-16. Pubmed link to this paper.
Painter HJ, Carrasquilla M, Llinás M. “Capturing in vivo RNA transcriptional dynamics from the malaria parasite Plasmodium falciparum.” Genome Research. 2017 Apr 17. doi: 10.1101/gr.217356.116. Pubmed link to this paper.
Kafsack BFC, Rovira-Graells N, Clark TG, Bancells C, Crowley VM, Campino SG, Williams AE, Drought LG, Kwiatkowski DP, Baker DA, Cortés A, Llinás M. “A transcriptional switch underlies commitment to sexual development in malaria parasites.” Nature. 2014 Mar 13;507(7491):248-52. Pubmed link to this paper.
Pino P, Sebastian S, Kim A, Bush E, Brochet M, Kozlowski E, Llinás M, Billker O, Soldati D. “A tetracycline-repressible transactivator system to study genes essential to Plasmodium bergheidevelopment.” Cell Host & Microbe. 2012, 12 (6). Pubmed link to this paper
Plata G, Hsiao T-L, Olszewski KL, Llinás M, Vitkup D. “Reconstruction and flux-balance analysis of the Plasmodium falciparum metabolic network.” Molecular Systems Biology. 2010 6: 408. Pubmed link to this paper.
Campbell TL, De Silva EK, Olszewski KL, Elemento O, Llinás M. “Identification and genome-wide mapping of DNA binding specificities for the ApiAP2 family of regulators from the malaria parasite.” PLoS Pathogens. (2010) 6 (10). Pubmed link to this paper
Lindner SE, De Silva EK, Keck JL, Llinás M. “Structural Determinants of DNA Binding by a P. falciparum ApiAP2 Transcriptional Regulator.” J Mol Biol. 2010 395 (3). Pubmed link to this paper
Olszewski KL, Morrisey JM, Wilinski D, Burns JM, Vaidya AB, Rabinowitz JD, Llinás M. “Host-parasite Interactions Revealed by Plasmodium falciparum Metabolomics.” Cell Host & Microbe.2009 5 (2). Pubmed link to this paper
Llinás M and Bozdech Z, Pulliam BL, Wong ED, Zhu J, DeRisi JL. “The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum.” PLoS Biology. 2003 1 (1). Pubmed link to this paper