Molecular Biology Faculty
|Llinás Research Lab|
Carl Icahn Lab, 246
Lab (609) 258-9496
Molecular mechanisms of the malaria parasite Plasmodium falciparum
Malaria is one of the most ancient unresolved afflictions of humankind. This disease is caused by the Plasmodium parasite, of which Plasmodium falciparum is the deadliest species. Malaria continues to affect upwards of half a billion people worldwide and kills over 1.5 million persons annually, mostly young children in sub-Saharan Africa. While there have been major global efforts to eliminate this disease, including eradication of the mosquito vector as well as developing chemotherapeutics against the blood-stage parasite, this disease continues to present a major health burden in afflicted regions. A major concern is the rapid evolution of drug-resistant strains worldwide and the lack of new chemotherapeutics. The challenge we face is to identify and characterize novel drug targets for anti-malarial strategies.
The lifecycle of Plasmodium is highly complex and involves three major stages of development: 1) a mosquito stage, 2) a liver stage and 3) a red blood cell stage. During the repetitive 48-hour red blood cell stage, the parasite undergoes an incredibly diverse set of morphologically distinct developmental transitions, from red blood cell invasion to rupture and reinvasion. This cycle of infection and persistence in the red blood cells is responsible for the severe clinical outcome of the malaria disease.
Many of the fundamental aspects of this parasite and its relationship with the human host are not clearly understood. One of the main questions is understanding how the parasite controls its developmental progression within the red blood cell. What are the factors that it uses to control this development? How does it interact with and sense the extracellular environment? And how are alternative developmental programs realized by the parasite?
We are using a combination of genomics, biochemistry, and bioinformatics to address these issues with the goal of devising ways to disrupt the parasite lifecycle.
Transcriptional regulation in P. falciparum
The whole genome sequence of Plasmodium falciparum has recently been completed, revealing several unique features. This landmark has opened up exciting possibilities for novel approaches toward research in malaria and has led to an increased fervor to identify new potential targets for anti-malarial drugs and vaccines. Surprisingly, approximately 60% of the predicted open reading frames share no homology to those of any other known organism. In addition, the genomic nucleotide composition is over 80% A/T in coding regions and 90% in non-coding regions. Lastly, there is a paucity of regulatory transcription factors in comparison to other eukaryotic organisms. This suggests that regulation of gene expression throughout development may be governed by a small number of factors or by a novel mechanism. In support of this hypothesis, the global transcriptional program of the intraerythrocytic developmental cycle (IDC) exhibits a continuous cascade of gene expression.
My lab is applying a variety of whole-genome approaches to investigate the molecular mechanism of transcriptional regulation in P. falciparum. Using DNA microarrays to analyze changes in transcription due to environmental perturbations such as drug challenge, we hope to define specific pathways which are triggered by these stimuli. We will also apply a variety of biochemical approaches to identify the factors involved in these regulatory processes. Further mutational analysis and knockouts of these factors coupled with structural analyses will help to unravel the details of gene regulation and the effects on genome wide transcription in vivo. We would also like to understand the interaction between these factors and the parasite genome. By using chromatin immunoprecipitation (ChIP-chip) methods, we plan to identify the cognate recognition sequences used by these putative regulatory proteins as well as their genome-wide distribution.
P. falciparum genome exploration
Although over half of the predicted P. falciparum genes have no known homologue in any other species, we have found that most of these novel genes are expressed, and thus presumably utilized, throughout the blood stage developmental cycle. While occasionally these unknown genes have homology to other P. falciparum sequences, often this is not the case. We can exploit the IDC transcriptome data to identify gene subgroups with similar expression profiles and potentially similar functions. In addition, we are now developing computational tools to analyze these novel or "hypothetical" protein sequences more rigorously incorporating all available sequence information from other Plasmodium species. This comparative analysis between genomes not only detects inter-species variability, but also identifies essential portions of the genome through well-conserved regions. Lastly, we are interested in the large portion of the P. falciparum genome that is comprised of genes rich in repetitive sequences. It is well established that these repeats vary from strain to strain and are especially common to antigens involved in host immune evasion. However, it remains to be determined whether these repeats encode regions of flexibility, interaction domains for other proteins, or simply share common structural motifs.
Kafsack BF, Painter HJ, Llinás M. (2012) New Agilent platform DNA microarrays for transcriptome analysis of Plasmodium falciparum and Plasmodium berghei for the malaria research community. Malar J. 11: 187. Pubmed
Olszewski KL, Llinás M. (2013) Extraction of hydrophilic metabolites from Plasmodium falciparum-infected erythrocytes for metabolomic analysis. Methods Mol Biol. 923: 259-266. Pubmed
Painter HJ, Altenhofen LM, Kafsack BF, Llinás M. (2013) Whole-genome analysis of Plasmodium spp. Utilizing a new Agilent Technologies DNA microarray platform. Methods Mol Biol. 923: 213-219. Pubmed
Painter HJ, Llinás M. (2012) Comment on "emerging functions of transcription factors in malaria parasite". J Biomed Biotechnol. 2012: 754054. PubMed
Klein EY, Lewis IA, Jung C, Llinás M, Levin SA. (2012) Relationship between treatment-seeking behaviour and artemisinin drug quality in Ghana. Malar J. 11:110. PubMed
Ochoa A, Llinas M, Singh M. (2011) Using context to improve protein domain identification. BMC Bioinformatics. 12: 90. PubMed
Painter HJ, Campbell TL, Llinás M. (2010) The Apicomplexan AP2 family: Integral factors regulating Plasmodium development. Mol Biochem Parasitol. 176: 1-7. PubMed
Campbell TL, De Silva EK, Olszewski KL, Elemento O, Llinás M. (2010) Identification and genome-wide prediction of DNA binding specificities for the ApiAP2 family of regulators from the malaria parasite. PLoS Pathog. 6: e1001165. PubMed
Olszewski KL, Llinás M. (2010) Central carbon metabolism of Plasmodium parasites. Mol Biochem Parasitol. 175: 95-103. PubMed
Olszewski KL, Mather MW, Morrisey JM, Garcia BA, Vaidya AB, Rabinowitz JD, Llinás M. (2010) Branched tricarboxylic acid metabolism in Plasmodium falciparum. Nature. 466: 774-778. PubMed
Kafsack BF, Llinás M. (2010) Eating at the table of another: metabolomics of host-parasite interactions. Cell Host Microbe. 7: 90-99. PubMed
Olszewski KL, Morrisey JM, Wilinski D, Burns JM, Vaidya AB, Rabinowitz JD, Llinás M. (2009) Host-parasite interactions revealed by Plasmodium falciparum metabolomics. Cell Host Microbe. 5: 191-199. PubMed
Llinás M, Deitsch KW, Voss TS. (2008) Plasmodium gene regulation: far more to factor in. Trends Parasitol. 24: 551-556. PubMed
De Silva EK, Gehrke AR, Olszewski K, León I, Chahal JS, Bulyk ML, Llinás M. (2008) Specific DNA-binding by apicomplexan AP2 transcription factors. Proc Natl Acad Sci USA. 105: 8393-8398. PubMed
Llinás M, Bozdech Z, Wong ED, Adai AT and DeRisi JL (2006). Comparative whole genome transcriptome analysis of three Plasmodium falciparum strains. Nucleic Acids Res 34: 1166-1173. PubMed
Llinás M, del Portillo HA (2005). Mining the malaria transcriptome. Trends Parasitol 21: 350-352. PubMed
Llinás M and DeRisi JL (2004). Pernicious plans revealed: Plasmodium falciparum genome wide expression analysis. Curr Opin Microbiol 7: 382-387. PubMed
Bozdech Z, Llinás M, Pulliam BL, Wong ED, Zhu J, DeRisi JL (2003). The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 1: E5. PubMed
Llinás M, Gillespie B, Dahlquist FW and Marqusee S (1999). The energetics of T4 lysozyme reveal a hierarchy of conformations. Nat Struct Biol 6: 1072-1078. PubMed
Liu H, Farr-Jones S, Ulyanov NB, Llinas M, Marqusee S, Groth D, Cohen FE, Prusiner SB, James TL (1999). Solution structure of Syrian hamster prion protein rPrP(90-231). Biochemistry 38: 5362-5377. PubMed
Llinás M and Marqusee S (1998). Subdomain interactions as a determinant in the folding and stability of T4 lysozyme. Protein Sci 7: 96-104. PubMed