Sebastian Lourido

Our lab is interested in the molecular events that enable apicomplexan parasites to remain widespread and deadly infectious agents. These single-celled eukaryotes comprise a phylum of organisms that parasitize diverse animal hosts. Many important human pathogens belong to this group, including the causative agents of malaria (Plasmodium spp.), cryptosporidiosis (Cryptosporidium spp.), and toxoplasmosis (Toxoplasma gondii). We use T. gondii to model features conserved throughout the phylum, such as their reliance on calcium signaling to regulate motility. We combine several approaches that span phospho-proteomics, chemical-genetics, and genome editing to investigate the unique biology of these organisms. Our work seeks to expand our understanding of eukaryotic diversity and identify specific features that can be targeted to treat parasite infections.

Model of the cell cycle of T. gondii illustrating key events in the transition from intracellular replication to extracellular motility (left), which are partly mediated by the activation of calcium signaling pathways (right).


Calcium Signaling
Changes in cytosolic calcium regulate eukaryotic cellular responses as diverse as membrane repair and muscle contraction. In apicomplexan parasites, calcium regulates motility in part through the regulation of adhesin exocytosis and myosin-motor function. We seek to understand the molecular details of these processes, examining both the events that lead to cytosolic calcium changes and the signaling events that follow them.
 
Protein Kinases
We are interested in how calcium signals are decoded by protein kinases, and in particular the role of calcium-dependent protein kinases (CDPKs) as the primary calcium-responsive kinases in parasites. Our work, and that of many others, has defined diverse roles for these kinases in regulating important events during the life cycle of apicomplexans. We have recently used alpaca-derived single-domain antibodies to probe the structure of CDPKs, defining a new mode of allosteric inhibition. We continue to develop biochemical methods to study these kinases, in addition to chemical-genetic approaches to study their function in vivo.
 
Chemical Genetics
We have engineered a panel of strains to study individual CDPKs. This approach relies on mutating the “gatekeeper” residue that restricts the depth of the ATP-binding pocket. This allows us to specifically inhibit or identify the targets of individual kinases. Using this approach, we have previously elucidated the distinct roles of TgCDPK1 and TgCDPK3 during the T. gondii lytic cycle. We continue to investigate the functions of these and other CDPKs, relying in part on phospho-proteomic methods to identify specific kinase targets.
 
Genome Engineering
Much of our work is made possible through a variety of genome-engineering methods. We are interested in developing new methods to enable efficient functional analysis of parasite genes and polymorphisms. Our lab was among the first to adapt CRISPR/Cas9 to engineer the T. gondii genome, and our plasmids are available to anyone through Addgene. We continue to improve these systems to enable genome-scale screening in parasites, which will allow­­ exploration of the multitude of apicomplexan genes with unknown functions.

Sidik SM, Huet D, Ganesan SM, Huynh MH, Wang T, Nasamu AS, Thiru P, Saeij JPJ, Carruthers VB, Niles JC, Lourido S. A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes. Cell. 2016 Sep;166(6):1423–1435.e12. PMID: 27594426

Sidik SM, Hortua Triana MA, Paul AS, El Bakkouri M, Hackett CG, Tran F, Westwood NJ, Hui R, Zuercher WJ, Duraisingh MT, Moreno SN, Lourido S. Using a genetically encoded sensor to identify inhibitors of Toxoplasma gondii Ca2+ signaling. J Biol Chem. 2016 Apr 29; 291(18):9566-80. PMID: 26933036

Rothenberg DA, Gordon EA, White FM, Lourido S. Identification of Direct Kinase Substrates Using Analogue-Sensitive Alleles. Methods Mol Biol. 2016; 1355:71-84. PMID: 26584919

Ingram JR, Knockenhauer KE, Markus BM, Mandelbaum J, Ramek A, Shan Y, Shaw DE, Schwartz TU, Ploegh HL, Lourido S. Allosteric activation of apicomplexan calcium-dependent protein kinases. Proc Natl Acad Sci U S A. 2015 Sep 8; 112(36):E4975-84. PMID: 26305940

Lourido S, Moreno S. The calcium signaling toolkit of the apicomplexan parasites Toxoplasma gondii and Plasmodium spp. Cell Calcium. 2015 Mar; 57(3):186-93. Review. PMID: 25605521

Sidik SM, Hackett CG, Tran F, Westwood NJ, Lourido S. Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS One. 2014 Jun 27; 9(6):e100450. PMID: 24971596

Lourido S, Jeschke GR, Turk BE, Sibley LD. Exploiting the unique ATP-binding pocket of Toxoplasma calcium-dependent protein kinase 1 to identify its substrates. ACS Chem Biol. 2013; 8(6):1155-62. PMID: 23530747

Lourido S, Zhang C, Lopez MS, Tang K, Barks J, Wang Q, Wildman SA, Shokat KM, Sibley LD. Optimizing small molecule inhibitors of calcium-dependent protein kinase 1 to prevent infection by Toxoplasma gondii. J Med Chem. 2013 Apr 11; 56(7):3068-77. PMID: 23470217

Lourido S, Tang K, Sibley LD. Distinct signalling pathways control Toxoplasma egress and host-cell invasion. EMBO J. 2012 Dec 12; 31(24):4524-34. PMID: 23149386

Lourido S, Shuman J, Zhang C, Shokat KM, Hui R, Sibley LD. Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature. 2010 May 20; 465(7296):359-62. PMID: 20485436

Billker O, Lourido S, Sibley LD. Calcium-dependent signaling and kinases in apicomplexan parasites. Cell Host Microbe. 2009 Jun 18; 5(6):612-22. Review. PMID: 19527888