Reconstruction and systems analysis of metabolism in apicomplexan parasites Toxoplasma gondii and Plasmodium falciparum

Understanding of metabolism in disease-causing microorganisms promotes drug design through the identification of the enzymes whose activity is indispensable for important cellular functions of the pathogens. Nowadays such understanding arises from experimental as well as computational studies. These two approaches, long considered as rather orthogonal, in recent years began to converge and form a new field, where they are utilized as complementary. In this thesis I present my endeavors in bringing closer the fields of infection and systems biology with a particular focus on large-scale metabolic models and their analysis. Integrative, interdisciplinary nature of my project also included multiple experimental inputs as well as original experimental efforts on investigating model-derived hypotheses. In the scope of this thesis I explored metabolism of two of the most experimentally amenable apicomplexan species â human parasites Plasmodium falciparum and Toxoplasma gondii. As a foundation for the studies included in this thesis I used standard as well as recently developed computational algorithms, existing experimental datasets and innovative context- specific assumptions. I produced an extensive survey of the modeling efforts previously applied for studying metabolism of P. falciparum and available large-scale experimental datasets in comparison with the similar efforts made in other species. Further, I curated an existing model of metabolism in Plasmodium falciparum with respect to an up-to-date primary literature on metabolism of the parasite and addressed several important assumptions implicitly made in this model. Using a state-of-the-art approach, I reconstructed de novo a comprehensive metabolic model of T. gondii, and performed an extensive computational analysis to explore its metabolic needs and capabilities. I identified and classified the minimal set of substrates the parasite utilizes for growth, along with the genes and pairs of genes that are essential for cellular functions such as growth and energy metabolism. Subsequently, several of the model-driven hypotheses were confirmed experimentally, while for validation of the majority of the computational predictions forthcoming high-throughput approaches shall be used. Every confirmed hypothesis expands the scope of our knowledge on peculiarities of metabolism in apicomplexan parasites and hence can serve as an input for the pipeline of developing novel medicines

Analysis and Design of CMOS Radio-Frequency Power Amplifiers

The continuous advancement of semiconductor technologies, especially CMOS technology, has enabled exponential growth of the wireless communication industry. This explosive growth in turn has completely changed people’s lives. The CMOS feature size scale down greatly benefits digital logic integrations, which result in more powerful, versatile, and economical digital signal processing. Further research and development has pushed analog, mixed-signal, and even radio-frequency (RF) circuit blocks to be implemented and integrated in CMOS. Future generations of wireless communication call for even further level of integration, and as of now, the only circuit block that is rarely integrated in CMOS along with other parts of the system is the power amplifier (PA). Due to the fact that the PA in a wireless communication system is the most power-hungry circuit block, the integration of RF PA in CMOS would potentially not only save the cost of the wireless communication system real estate, but also reduce power consumption since die-to-die connection loss can be eliminated. RF PA design involves handling large amounts of voltage and current at the radio frequencies, which in the present wireless communication standards are in the range of giga-hertz. Therefore, a good understanding of many aspects related to RF PA design is necessary. Theoretical analysis of the communication system, nonlinear effects of the PA, as well as the impedance matching network is systematically presented. The analysis of the nonlinear effects proposes a formal mathematical description of the multitone nonlinearity, and through its relationship with two-tone test, the proposed PA design methodology would greatly reduce the design time while improving the design accuracy. A thorough analysis of the available architecture and design techniques for efficiency and linearity enhancement of RF PA shows that despite tremendous amounts of research and development into this topic, the fundamental tradeoff between the two still limits the RF PA implementation largely within SiGe, GaAs, and InP technologies. A RF PA for Wideband Code-Division Multiple Access (WCDMA) application standard is proposed, designed, and implemented in CMOS that demonstrates the proposed segmentation technique that resolved the main tradeoff between power efficiency and linearity. The innovative architecture developed in this work is not limited to applications in the WCDMA communication protocol or the CMOS technology, although CMOS implementation would take advantage of the readily available digital resources

Functional genomics of Plasmodium falciparum using metabolic modelling and analysis

Plasmodium falciparum is an obligate intracellular parasite and the leading cause of severe malaria responsible for tremendous morbidity and mortality particularly in sub-Saharan Africa. Successful completion of the P. falciparum genome sequencing project in 2002 provided a comprehensive foundation for functional genomic studies on this pathogen in the following decade. Over this period, a large spectrum of experimental approaches has been deployed to improve and expand the scope of functionally annotated genes. Meanwhile, rapidly evolving methods of systems biology have also begun to contribute to a more global understanding of various aspects of the biology and pathogenesis of malaria. Herein we provide an overview on metabolic modelling, which has the capability to integrate information from functional genomics studies in P. falciparum and guide future malaria research efforts towards the identification of novel candidate drug targets

Metabolic Needs and Capabilities of Toxoplasma gondii through Combined Computational and Experimental Analysis

Toxoplasma gondii is a human pathogen prevalent worldwide that poses a challenging and unmet need for novel treatment of toxoplasmosis. Using a semi-automated reconstruction algorithm, we reconstructed a genome-scale metabolic model, ToxoNet1. The reconstruction process and flux-balance analysis of the model offer a systematic overview of the metabolic capabilities of this parasite. Using ToxoNet1 we have identified significant gaps in the current knowledge of Toxoplasma metabolic pathways and have clarified its minimal nutritional requirements for replication. By probing the model via metabolic tasks, we have further defined sets of alternative precursors necessary for parasite growth. Within a human host cell environment, ToxoNet1 predicts a minimal set of 53 enzyme-coding genes and 76 reactions to be essential for parasite replication. Double-gene-essentiality analysis identified 20 pairs of genes for which simultaneous deletion is deleterious. To validate several predictions of ToxoNet1 we have performed experimental analyses of cytosolic acetyl-CoA biosynthesis. ATP-citrate lyase and acetyl-CoA synthase were localised and their corresponding genes disrupted, establishing that each of these enzymes is dispensable for the growth of T. gondii, however together they make a synthetic lethal pair

Nutritional requirements of <i>P</i>. <i>falciparum</i> concerning essential backbone moieties.

<p>Note: the presence of orthophosphate and S-adenosylmethioninamine in the same medium is not allowed. When <i>P</i>. <i>falciparum</i> grows on orthophosphate and S-adenosylmethioninamine as the only sources of phosphate and purines, it cannot synthesize enough ATP. The ATP limitation impedes the production of other phosphorylated nucleotides, sugar nucleotides, sphingomyelin and phospholipids in the stoichiometrically required amounts. As suggested throughout the manuscript, these metabolic processes are thermodynamically dependent (more details in the <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005397#pcbi.1005397.s001" target="_blank">S1 Methods</a>).</p

Generation of an inducible ACL knockdown in ACSko parasites.

<p>(A) Schematic representation of the U1 snRNP-mediated ACL gene silencing with Cre-recombinase dependent positioning of U1 in Ku80ko wildtype and ACSko parasites. (B) PCRs performed on genomic DNA extracted from Ku80ko, ACL-lox, ACSko/ACL-lox validating integration of the pKI-ACL-3TyLox3’UTRLoxU1 construct to knock down ACL in the different strains. The sequences of the primers can be found in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004261#pcbi.1004261.s008" target="_blank">S7 Table (C and D)</a>. Immuno-blot of total lysates from ACL-lox, ACSko/ACL-lox where ACL-lox was integrated in the ACSko strain or ACS was knocked out in ACL-lox. Both independent lines show increased levels of ACL when ACS is absent. Western blot was performed using anti-Ty antibodies. Anti-TgProfilin (Prf) represents a loading control.</p

Gene essentiality predictions of ToxoNet 1 and available literature evidence.

<p>(NA denotes the cases when neither supporting nor contradicting literature reference could be found). Gene essentiality in <i>P</i>. <i>falciparum</i> is based on the Supplementary Table 1 of the review manuscript [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004261#pcbi.1004261.ref038" target="_blank">38</a>].</p><p>Gene essentiality predictions of ToxoNet 1 and available literature evidence.</p

Both ACS and ACL are dispensable in the tachyzoite stage of <i>T</i>. <i>gondii</i>.

<p>(A) Schematic representation of the two pathways to produce acetyl-CoA in the cytosol of <i>T</i>. <i>gondii</i>. Abbreviations: AcCoA, acetyl-CoA; α-KG, α-ketoglutarate; Cit, citrate; Glc, glucose; Lac, lactate; Mal, malate; OAA, oxaloacetic acid; Pyr, pyruvate; Suc, succinate. Enzymes in red: ACL, ATP-citrate lyase; ACS, Acetyl-CoA synthetase. (B) Scheme of the knock-in strategy used to introduce a 3Ty-tag in the endogenous loci of ACS, ACL and AT1. (C) Localization of endogenous ACS, ACL and AT1 C-terminally Ty-tagged (ACS-3Ty, ACL-3Ty and AT1-3Ty) in the cytoplasm, cytosol and endoplasmic reticulum respectively of intracellular parasites using anti-Ty as well as anti-GAP45 that stains the periphery and DAPI which stains the nucleus of the parasite. (D) Immuno-blot of total lysates from Ku80ko parasites expressing the C-terminally Ty-tagged endogenous ACS, ACL and AT1 proteins by Western blot using anti-Ty antibodies. Anti-Profilin (Prf) represents a loading control. (E) Schematic representation of the direct knockout strategy by double homologous recombination where ACS was replaced by the chloramphenicol resistance cassette and ACL by the HXGPRT selection cassette. The position of the primers used to confirm the integration and the length of the PCR products are indicated. PCRs performed on genomic DNA extracted from Ku80ko, ACSko and ACLko strains confirm the integration of the selection cassette and loss of the corresponding gene locus. The sequences of the primers can be found in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004261#pcbi.1004261.s008" target="_blank">S7 Table</a>. (F) Plaque assays performed with Ku80ko, ACSko and ACLko parasite lines fixed after 7 days. No significant defect in the lytic cycle could be observed. (G) Intracellular growth assay performed on Ku80ko, ACSko and ACLko strains by determining the number of parasites per vacuole 24h post infection. Data are represented as mean ± SD from 3 biological replicates.</p

Reconstruction workflow for ToxoNet1 summarized as a flowchart illustration.

<p>Reconstruction workflow for ToxoNet1 summarized as a flowchart illustration.</p

Comparison of the number of metabolites, reactions and genes between the models for <i>T</i>. <i>gondii</i> and <i>P</i>. <i>falciparum</i>.

<p>Abbreviations: e—extracellular space, c—cytosol, m—mitochondrion, a—apicoplast, n—nucleus, er—endoplasmic reticulum, g—Golgi complex, v—digestive vacuole, im—mitochondrial intermembrane space.</p><p>Comparison of the number of metabolites, reactions and genes between the models for <i>T</i>. <i>gondii</i> and <i>P</i>. <i>falciparum</i>.</p