Versatile control of Plasmodium falciparum gene expression with an inducible protein–RNA interaction

The available tools for conditional gene expression in Plasmodium falciparum are limited. Here, to enable reliable control of target gene expression, we build a system to efficiently modulate translation. We overcame several problems associated with other approaches for regulating gene expression in P. falciparum. Specifically, our system functions predictably across several native and engineered promoter contexts, and affords control over reporter and native parasite proteins irrespective of their subcellular compartmentalization. Induction and repression of gene expression are rapid, homogeneous and stable over prolonged periods. To demonstrate practical application of our system, we used it to reveal direct links between antimalarial drugs and their native parasite molecular target. This is an important outcome given the rapid spread of resistance, and intensified efforts to efficiently discover and optimize new antimalarial drugs. Overall, the studies presented highlight the utility of our system for broadly controlling gene expression and performing functional genetics in P. falciparum.National Institutes of Health (U.S.) (Health Director's New Innovator Award 1DP2OD007124)Bill & Melinda Gates Foundation (Grand Challenges Explorations Initiative OPP1069759)National Institute of Environmental Health Sciences (Predoctoral Training Grant 5-T32-ES007020)National Institute of General Medical Sciences (U.S.) (Biotechnology Training Grant 5-T32-GM08334)Thomas and Stacey Siebel FoundationMIT Start-up Fund

A microfabricated deformability-based flow cytometer with application to malaria

Malaria resulting from Plasmodium falciparum infection is a major cause of human suffering and mortality. Red blood cell (RBC) deformability plays a major role in the pathogenesis of malaria. Here we introduce an automated microfabricated “deformability cytometer” that measures dynamic mechanical responses of 10[superscript 3] to 10[superscript 4] individual RBCs in a cell population. Fluorescence measurements of each RBC are simultaneously acquired, resulting in a population-based correlation between biochemical properties, such as cell surface markers, and dynamic mechanical deformability. This device is especially applicable to heterogeneous cell populations. We demonstrate its ability to mechanically characterize a small number of P. falciparum-infected (ring stage) RBCs in a large population of uninfected RBCs. Furthermore, we are able to infer quantitative mechanical properties of individual RBCs from the observed dynamic behavior through a dissipative particle dynamics (DPD) model. These methods collectively provide a systematic approach to characterize the biomechanical properties of cells in a high-throughput manner.National Institutes of Health (U.S.) (Grant R01 HL094270-01A1)National Institutes of Health (U.S.) (Grant 1-R01-GM076689-01)Singapore-MIT Alliance for Research and Technology Cente

Single T Cell Sequencing Demonstrates the Functional Role alpha beta TCR Pairing in Cell Lineage and Antigen Specificity

Although structural studies of individual T cell receptors (TCRs) have revealed important roles for both the alpha and beta chain in directing MHC and antigen recognition, repertoire-level immunogenomic analyses have historically examined the beta chain alone. To determine the amount of useful information about TCR repertoire function encoded within alpha beta pairings, we analyzed paired TCR sequences from nearly 100,000 unique CD4+ and CD8+ T cells captured using two different high-throughput, single-cell sequencing approaches. Our results demonstrate little overlap in the healthy CD4+ and CD8+ repertoires, with shared TCR sequences possessing significantly shorter CDR3 sequences corresponding to higher generation probabilities. We further utilized tools from information theory and machine learning to show that while alpha and beta chains are only weakly associated with lineage, of pairings appear to synergistically drive TCR-MHC interactions. V alpha beta gene pairings were found to be the TCR feature most informative of T cell lineage, supporting the existence of germline-encoded paired alpha beta TCR-MHC interaction motifs. Finally, annotating our TCR pairs using a database of sequences with known antigen specificities, we demonstrate that approximately a third of the T cells possess alpha and beta chains that each recognize different known antigens, suggesting that alpha beta pairing is critical for the accurate inference of repertoire functionality. Together, these findings provide biological insight into the functional implications of alpha beta pairing and highlight the utility of single-cell sequencing in immunogenomics

Direct and specific chemical control of eukaryotic translation with a synthetic RNA–protein interaction

Sequence-specific RNA–protein interactions, though commonly used in biological systems to regulate translation, are challenging to selectively modulate. Here, we demonstrate the use of a chemically-inducible RNA–protein interaction to regulate eukaryotic translation. By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5′-untranslated region (5′-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5′-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA–TetR interactions. Using a reverse TetR variant, we illustrate the potential for expanding the regulatory properties of the system through protein engineering strategies

MalDA, Accelerating Malaria Drug Discovery

© 2021 The Authors The Malaria Drug Accelerator (MalDA) is a consortium of 15 leading scientific laboratories. The aim of MalDA is to improve and accelerate the early antimalarial drug discovery process by identifying new, essential, druggable targets. In addition, it seeks to produce early lead inhibitors that may be advanced into drug candidates suitable for preclinical development and subsequent clinical testing in humans. By sharing resources, including expertise, knowledge, materials, and reagents, the consortium strives to eliminate the structural barriers often encountered in the drug discovery process. Here we discuss the mission of the consortium and its scientific achievements, including the identification of new chemically and biologically validated targets, as well as future scientific directions

Replication and Recombination Factors Contributing to Recombination-Dependent Bypass of DNA Lesions by Template Switch

Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to template switch in response to DNA damage and to distinguish this process from other recombination-mediated processes promoting DNA repair

Engineering control of eukaryotic translation with application to the malaria parasite Plasmodium falciparum

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014.Cataloged from PDF version of thesis.Includes bibliographical references (pages 123-130).Experimenter control of target gene expression is a fundamental component of molecular biology research. In many systems, tools exist that allow generalizable control of gene expression at the transcriptional or post-transcriptional level. Plasmodium falciparum, the protozoan parasite responsible for the majority of death and sickness due to malaria, remains challenging to manipulate in the laboratory. No robust and generalizable tool for gene expression control has been developed in the parasite. To address this need, we engineered a new system for control of protein translation in eukarvotes, and applied it to P. falciparum. This system is based on the ligand-regulated interaction between an RNA aptamers and the TetR-repressor protein. Although such protein-RNA interactions are abundant in nature and are known to effectively mediate control of gene expression, our system is unique in its direct modulation by an exogenous chemical. By genetically encoding TetR-binding RNA aptamers in the 5' untranslated region (5'UTR) of an mRNA, translation of a downstream coding sequence is repressed by TetR in vivo and induced upon adding a non-toxic tetracycline analog. We first define the system's component molecular interactions in vitro, followed by optimization of the constituent parts for convenience and performance. We then further optimize the system and validate its performance in two model systems, the budding yeast Saccharomvces cerevisiae and cell-free rabbit reticulocyte extracts. We show the broad utility of the system in P. falciparum for controlling expression of reporter and endogenous proteins trafficked to a variety of subcellular compartments. Induction and repression are rapid and homogeneous across the cell population. Placing a drug resistance determinant tinder inducible control, we are able to modulate P. falciparum drug sensitivity, demonstrating the usefulness of the system for controlling relevant parasite biology. In the process of constructing and validating a novel tool for gene expression in P. falciparum. we built a new series of gene expression vectors for molecular biology work in the parasite. In addition to developing optimized protocols for plasmid construction, we built a standardized, sequence-defined family of plasmids for malaria research. In all, we present a generalizable, well-defined toolkit for genetic programming of P. falciparum.by Stephen J. Goldfless.Ph. D

Synthetic RNA–protein modules integrated with native translation mechanisms to control gene expression in malaria parasites

Synthetic posttranscriptional regulation of gene expression is important for understanding fundamental biology and programming new cellular processes in synthetic biology. Previous strategies for regulating translation in eukaryotes have focused on disrupting individual steps in translation, including initiation and mRNA cleavage. In emphasizing modularity and cross-organism functionality, these systems are designed to operate orthogonally to native control mechanisms. Here we introduce a broadly applicable strategy for robustly controlling protein translation by integrating synthetic translational control via a small-molecule-regulated RNA–protein module with native mechanisms that simultaneously regulate multiple facets of cellular RNA fate. We demonstrate that this strategy reduces ‘leakiness’ to improve overall expression dynamic range, and can be implemented without sacrificing modularity and cross-organism functionality. We illustrate this in Saccharomyces cerevisae and the non-model human malarial parasite, Plasmodium falciparum. Given the limited functional genetics toolkit available for P. falciparum, we establish the utility of this strategy for defining essential genes.Massachusetts Institute of Technology (MIT startup funds)Thomas and Stacey Siebel Foundation (Award)National Institute of General Medical Sciences (U.S.) (Center for Integrative Synthetic Biology Grant (P50 GM098792))National Institute of Environmental Health Sciences (Predoctoral Training Grant (5-T32-ES007020))Bill & Melinda Gates Foundation (Grand Challenges Explorations initiative (OPP1069759))National Institutes of Health (U.S.) (NIH Director’s New Innovator Award (1DP2OD007124)