State College, Pennsylvania 16803
Understanding the molecular mechanisms of gene regulation and metabolism in the malaria parasite Plasmodium falciparum using functional genomics and metabolomics.
- B.S. Chemistry Carnegie Mellon University (1992)
- Ph.D. University of California, Berkeley (1999)
Understanding the molecular mechanisms of gene regulation and metabolism in the malaria parasite Plasmodium falciparum using functional genomics and metabolomics.
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.
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.
Cobbold SA, Santos JM, Ochoa A, Perlman DH, Llinás M. Proteome-wide analysis reveals widespread lysine acetylation of major protein complexes in the malaria parasite. 2016. Scientific Reports, 19722. doi: 10.1038/srep19722.
Adjalley SH, Scanfeld D, Kozlowski E, Llinás M, Fidock DA. Genome-wide transcriptome profiling reveals functional networks involving the Plasmodium falciparum drug resistance transporters PfCRT and PfMDR1. BMC Genomics, 2015. 21;16(1):1090.
Cobbold SA, Llinás M, Kirk K. Sequestration and metabolism of host cell arginine by the intraerythrocytic malaria parasita Plasmodium falciparum. Cellular Microbiology 2015. 3;doi: 10.111/cmi.12552
Ochoa A, Sotrey JD, Llinás M, Singh M. Beyond the E-Value: Stratified Statistics for Protein Domain Prediction. PLoS Computational Biology. 2015. 11(11):31004509
Pulcini S, Staines HM, Lee AH, Shafic SH, Bouyer G, Moore DM, Daley DA, Joke MJ, Altenhofen LM, Painter HJ, Muy J, Ferguson DH, Llinás M, Martin RE, Fidock DA, Cooper RA, Krishna S. Mutation in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities. Scientific Reports. 2015. 5:14552. doi: 10.1038/srep14552
Josling GA, Llinás M. Sexual development in Plasmodium parasites: knowing when it’s time to commit. Nature Reviews Microbiology 2015 Aug 14;13(9):573-87. Cell Reports. 2015 Apr 7;11(1):164-74. doi: 10.1016/j.celrep.2015.03.011. Epub 2015 Apr 2. (Supplement)
Eshar S, Altenhofen L, Rabner A, Ross P, Fastman Y, Mandel-Gutfreund Y, Karni R, Llinás M, Dzikowski R. PfSR1 controls alternative splicing and steady-state RNA levels in Plasmodium falciparum through preferential recognition of specific RNA motifs.” Molecular Microbiology. 2015 Jun;96(6):1283-97. doi: 10.1111/mmi.13007. Epub 2015 Apr 16. (Supplement)
Lisewski AM, Quiros JP, Ng CL, Adikesavan AK, Miura K, Putluri N, Eastman RT, Scanfeld D, Regenbogen SJ, Altenhofen L, Llinás M, Sreekumar A, Long C, Fidock DA, Lichtarge O. Supergenomic Network Compression and the Discovery of EXP1 as a Glutathione Transferase Inhibited by Artesunate. Cell. 2014 Aug 14;158(4):916-28. doi: 10.1016/j.cell.2014.07.011.
Coleman BI, Skillman KM, Jiang RH, Childs LM, Altenhofen LM, Ganter M, Leung Y, Goldowitz I, Kafsack BF, Marti M, Llinás M, Buckee CO, Duraisingh MT. A Plasmodium falciparum Histone Deacetylase Regulates Antigenic Variation and Gametocyte Conversion. Cell Host & Microbe. 2014 Aug 13;16(2):177-86. doi: 10.1016/j.chom.2014.06.014.
Oberstaller J, Pumpalova Y, Schieler A, Llinás M, Kissinger JC. The Cryptosporidium parvum ApiAP2 gene family: insights into the evolution of apicomplexan AP2 regulatory systems.” Nucleic Acids Research. 2014 June 23. pii: gku500.
O’Hara JK, Kerwin LJ, Cobbold SA, Tai J, Bedell TA, Reider PJ, Llinás M. Targeting NAD+ metabolism in the human nalaria parasite Plasmodium falciparum.” PLoS ONE. 2014 April; 9(4):e94061. (Supplement)
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. (Supplement)
Sinha A, Hughes KR, Modrzynska KK, Otto TD, Pfander C, Dickens NJ, Religa AA, Bushell E, Graham AL, Cameron R, Kafsack BFC, Williams AE, Llinás M, Berriman M, Billker O, Waters AP. A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium. Nature. 2014 Mar 13;507 (7491):253-7. (Supplement)
Lewis IA, Wacker M, Olszewski KL, Cobbold SA, Baska KS, Tan A, Ferdig MT, Llinás M. Metabolic QTL Analysis Links Chloroquine Resistance in Plasmodium falciparum to Impaired Hemoglobin Catabolism. PLoS Genetics. 2014. Jan;10(1):e1004085. Infection & Immunity. 2014 Jan;82(1): 140-51.
Klein EY, Graham AL, Llinás M, Levin S. Cross-reactive immune responses as primary drivers of malaria chronicity. Infection & Immunity. 2014 Jan;82(1): 140-51 (Supplement)
Cobbold SA, Vaughan AM, Lewis IA, Painter HJ, Camargo N, Perlman DH, Fishbaugher M, Healer J, Cowman AF, Kappe SH, Llinás M. Kinetic Flux Profiling Elucidates Two Independent Acetyl-CoA Biosynthetic Pathways in Plasmodium falciparum. Biol Chem. 2013 Dec 20; 288(51): 36338-50. (Supplement)
Caudy AA, Guan Y, et al. A new system for comparative functional genomics of Saccharomyces yeasts. Genetics. 2013 Sep; 195(1): 275-87.
Mira-Martínez S, Rovira-Graells N, Crowley VM, Altenhofen LM, Llinás M, Cortés A. Epigenetic switches in clag3 genes mediate blasticidin S resistance in malaria parasites. Cellular Microbiology. 2013 Nov;15(11): 1913-23.
Carter LM, Kafsack B, Llinás M, Pollitt L, Reece S. Stress and Sex in Malaria Parasites: Why Does Commitment Vary?” Evolution, Medicine, and Public Health. 2013 June 4; 135-147.
Radke JB, Lucas O, De Silva EK, Ma Y, Sullivan WJ Jr, Weiss LM, Llinás M, White MW. ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst. PNAS 2013 April 09.
Pino P, Sebastian S, Kim EA, Bush E, Brochet M, Volkmann K, Kozlowski E, Llinás M, Billker O, Soldati-Favre D. A tetracycline-repressible transactivator system to study essential genes in malaria parasites. Cell Host & Microbe 2012 Dec 13;12(6):824-34.
Babbitt SE, Altenhofen L, Cobbold SA, Istvan ES, Fennell C, Doerig C, Llinás M, Goldberg DE. Plasmodium falciparum responds to amino acid starvation by entering into a hibernatory state. PNAS 2012 Nov 20;109(47):E3278-87.
Painter HJ, Altenhofen LM, Kafsack BF, Llinás M. Whole-Genome Analysis of Plasmodium spp. Utilizing a New Agilent Technologies DNA Microarray Platform. Methods Mol Biol. 2012 923:213-9.
Olszewski KL, Llinás M. Extraction of Hydrophilic Metabolites from Plasmodium falciparum-Infected Erythrocytes for Metabolomic Analysis. Methods Mol Biol. 2012 923:259-66.
Straimer J, Lee MC, Lee AH, Zeitler B, Williams AE, Pearl JR, Zhang L, Rebar EJ, Gregory PD, Llinás M, Urnov FD, Fidock DA. Site-specific genome editing in Plasmodium falciparum using engineered zinc-finger nucleases. Nature Methods. 2012 Aug 2.
Kafsack BF, Painter HJ, Llinás M. New Agilent platform DNA microarrays for transcriptome analysis of Plasmodium falciparum and Plasmodium berghei for the malaria research community. Malaria Journal. 2012 Jun 8;11(1):187.
Painter HJ, Llinás M. Comment on “emerging functions of transcription factors in malaria parasite.” J Biomed Biotechnol. 2012 Epub 2012 May 20.
Klein EY, Lewis IA, Jung C, Llinás M, Levin SA. “Relationship between treatment-seeking behaviour and artemisinin drug quality in Ghana.” Malaria Journal. 2012 Apr 6;11:110.
Lindner SE, Llinás M, Keck JL, Kappe SH. The primase domain of PfPrex is a proteolytically matured, essential enzyme of the apicoplast. Molecular and Biochemical Parasitology. 2011 Dec;180(2):69-75.
Storm J, Aparicio I, Patzewitz EM, Olszewski K, Llinás M, Engel PC, Muller S. Plasmodium falciparum glutamate dehydrogenase a is dispensable and not a drug target during erythrocytic development. Malaria Journal. 2011 July 14; 10 (1): 193.
Ochoa A, Llinás M and Singh M.n Using context to improve protein domain identification. BMC Bioninformatics. 2011 March 31; 12 (1): 90.
Painter HJ, Campbell TL, and Llinás M. The Apicomplexan AP2 family: Integral factors regulating Plasmodium development. Molecular and Biochemical Parasitology. 2011 March; 176 (1) Epub Nov 2010.
Campbell TL, De Silva EK, Olszewski KL, Elemento O, Llinás M. Identification and genome-wide prediction of DNA binding specificities for the ApiAP2 family of regulators from the malaria parasite. PLoS Pathogens. 2010 October 28; 6 (10) (Supplementary text and figures file)
Helm S, Lehmann C, Nagel A, Stanway RR, Horstmann S, Llinás M, Heussler VT. Identification and characterization of a liver stage-specific promoter region of the malaria parasite Plasmodium. PLoS One. 2010 October 27; 5 (10).
Olszewski KL and Llinás M. Central carbon metabolism of malaria parasites.” Molecular and Biochemical Parasitology. 2010 September 14.
Plata G, Hsiao TL, Olszewski KL, Llinás M, Vitkup D. Reconstruction and flux-balance analysis of the Plasmodium falciparum metabolic network. Molecular Systems Biology. 2010 September 07; 6 (408).
Dowling DP, Ilies M, Olszewski KL, Portugal S, Mota MM, Llinás M, Christianson DW. Crystal structure of arginase from Plasmodium falciparum and implications for L-arginine depletion in malarial infection. Biochemistry 2010, July 6;49 (26), pp. 5600-8.
Otto DT, Wilinski D, Assefa S, Keane TM, Sarry LR, Böhme U, Lemieux J, Barrell B, Pain A, Berriman M, Newbold C, Llinás M. New insights into the blood stage transcriptome of Plasmodium falciparum using RNA-Seq. Molecular Microbiology 2010, April;67 (1), pp. 12-24.
Kafsack BF, Llinás M. Eating at the Table of Another: Metabolomics of Host-Parasite Interactions. Cell Host & Microbe. 2010, Feb 18; 7 (2) pp. 90-99.
Lindner SE, De Silva EK, Keck JL, Llinás M. Structural Determinants of DNA Binding by a P. falciparum ApiAP2 Transcriptional Regulator. Journal of Molecular Biology 2010, Feb 04;395 (3), pp. 558-67.
Howitt CA, Wilinski D, Llinás M, Templeton TJ, Dzikowski R, Deitsch KW. Clonally variant gene families in P. falciparum share a common activation factor. Molecular Microbiology 2009, Sept; 73 (6), pp. 1171-85.
Zawada RJ, Kwan P, Olszewski KL, Llinás M, Huang SG. Quantitative determination of urea concentrations in cell culture medium. Biochem Cell Biol. 2009 Jun;87(3):541-4.
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 Feb 19; 5(2), pp. 191-199. (Featured on the journal cover)
van Brummelen AC, Olszewski KL, Wilinski D, Llinás M, Louw AI, Birkholtz LM. Co-inhibition of Plasmodium falciparum S-adenosylmethionine decarboxylase/ornithine decarboxylase reveals perturbation-specific compensatory mechanisms by transcriptome, proteome and metabolome analyses. J Biol Chem. 2009 Feb 13;284(7), pp. 4635-46.
Llinás M., Deitsch KW., Voss TS. Plasmodium gene regulation: far more to factor in. Trends Parasitol. 2008. Dec;24(12), pp. 551-6.
Tamez PA., Bhattacharjee S., van Ooij C., Hiller NL., Llinás M., Balu B., Adams JH., Haldar K. An erythrocyte vesicle protein exported by the malaria parasite promotes tubovesicular lipid import from the host cell surface PLoS Pathog. 2008 Aug 8;4(8):e1000118.
De Silva EK., Gehrke AR., Olszewski K., León I, Chahal JS., Bulyk ML., Llinás M. Specific DNA-binding by Apicomplexan AP2 transcription factors. Proc Natl Acad Sci USA. 2008 Jun 17;105(24), pp. 8393-8.(See feature in Nature Reviews Microbiology)
Birkholtz L., van Brummelen AC., Clark K., Niemand J., Marechal E., Llinás M., Louw AI. Exploring functional genomics for drug target and therapeutics discovery in Plasmodia Acta Trop. 2008 Feb; 105(2), pp. 113-23.
Hu G., Llinás M., Li J., Preiser PR., Bozdech Z. Selection of long oligonucleotides for gene expression microarrays using weighted rank-sum strategy BMC Bioinformatics. 2007 Sep 19;8:350.
Deitsch K., Duraisingh M., Dzikowski R., Gunasekera A., Khan S., Le Roch K., Llinás M., Mair G., McGovern V., Roos D., Shock J., Sims J., Wiegand R., Winzeler E. Mechanisms of Gene Regulation in Plasmodium Am J Trop Med Hyg. 2007 Aug;77(2), pp. 201-8.
Llinás M., Bozdech Z., Wong ED., Adai, AT., DeRisi JL. Comparative whole genome transcriptome analysis of three Plasmodium falciparum strains Nucleic Acids Res. 2006 Feb 21;34(4), pp. 1166-73.
Llinás, M. and del Portillo HA. Mining the malaria transcriptome Trends Parasitol. 2005 Aug; 21(8), pp. 350-2.
Llinás, M. and DeRisi JL. Pernicious plans revealed: Plasmodium falciparum genome wide expression analysis Curr Opin Microbiol. 2004 Aug; 7(4), pp. 382-7.
Llinás M. and Bozdech Z., Pulliam BL., Wong ED., Zhu J., DeRisi JL. The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum PLoS Biol. 2003 Dec;3(12):e426.