Biology subjects

Llinas, M.

Publications and source records attributed to Llinas, M..

28 records · Page 2Linked to original sources

bioRxiv2020

Homeodomain protein 1 is an essential regulator of gene expression during sexual differentiation of malaria parasites

Transmission of Plasmodium falciparum and other malaria parasites requires their differentiation from asexual blood stages into gametocytes, the non-replicative sexual stage necessary for transmission to the mosquito vector. This transition involves changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. However, the genomes of malaria parasites have been noted for their dearth of transcriptional and chromatin regulators and the molecular mediators of these changes remain largely unknown. We recently identified HomeoDomain Protein 1 (HDP1) as a DNA-binding protein that enhances the expression of key genes that are critical for early sexual differentiation. The discovery of a homeodomain-like DNA-binding protein marks a new class of transcriptional regulator in malaria parasites outside of the better-characterized ApiAP2 family. In this study, we show that HDP1 facilitates the necessary upregulation of inner membrane complex components during early gametocytogenesis that gives P. falciparum gametocytes they characteristic shape and is required for gametocyte maturation and parasite transmission.

microbiology↗

bioRxiv2020

Atypical molecular basis for drug resistance to mitochondrial function inhibitors in Plasmodium falciparum

The continued emergence of drug-resistant Plasmodium falciparum parasites hinders global attempts to eradicate malaria, emphasizing the need to identify new antimalarial drugs. Attractive targets for chemotherapeutic intervention are the cytochrome (cyt) bc1 complex, which is an essential component of the mitochondrial electron transport chain (mtETC) necessary for ubiquinone recycling and mitochondrially localized dihydroorotate dehydrogenase (DHODH) critical for de novo pyrimidine synthesis. Despite the essentiality of this complex, resistance to a novel acridone class of compounds targeting cyt bc1 was readily attained, resulting in a parasite strain (SB1-A6) that was pan-resistant to both mtETC and DHODH inhibitors. Here we describe the molecular mechanism behind the resistance of the SB1-A6 parasite line which lacks the common cyt bc1 point mutations characteristic of resistance to mtETC inhibitors. Using Illumina whole-genome sequencing, we have identified both a copy number variation ([~]2x) and a single-nucleotide polymorphism (C276F) associated with pfdhodh in SB1-A6. We have characterized the role of both genetic lesions by mimicking the copy number variation via episomal expression of pfdhodh and introducing the identified SNP using CRISPR/Cas9 and assessed their contributions to drug resistance. Although both of these genetic polymorphisms have been previously identified as contributing to both DSM-1 (1) and atovaquone resistance (2, 3), SB1-A6 represents a unique genotype in which both alterations are present in a single line, suggesting that the combination contributes to the pan-resistant phenotype. This novel mechanism of resistance to mtETC inhibition has critical implications for the development of future drugs targeting the bc1 complex or de novo pyrimidine synthesis that could help guide future anti-malarial combination therapies and reduce the rapid development of drug resistance in the field.

microbiology↗

bioRxiv2020

Genetic ablation of the mitochondrial ribosome in Plasmodium falciparum sensitizes the human malaria parasite to antimalarial drugs targeting mitochondrial functions

The mitochondrion of malaria parasites contains clinically validated drug targets. Within Plasmodium spp., the mitochondrial DNA (mtDNA) is only 6 kb long, being the smallest mitochondrial genome among all eukaryotes. The mtDNA encodes only three proteins of the mitochondrial electron transport chain and [~] 27 small, fragmented rRNA genes in length of 22-195 nucleotides. The rRNA fragments are thought to form a mitochondrial ribosome (mitoribosome), together with ribosomal proteins imported from the cytosol. The mitoribosome of Plasmodium falciparum has been shown to be essential for maintenance of the mitochondrial membrane potential and parasite viability. However, the role of mitoribosomes in sustaining the metabolic status of the parasite mitochondrion remains unknown. Here, among the 14 annotated mitoribosomal proteins of the small subunit of P. falciparum, we verified the localization and tested the essentiality of three candidates (PfmtRPS12, PfmtRPS17, PfmtRPS18), employing a CRISPR/Cas9 mediated conditional knockdown tool. Using immuno-electron microscopy, we provided evidence that the mitoribosome is closely associated with the mitochondrial inner membrane in the parasite. Upon knockdown of the mitoribosome, the parasites became hypersensitive to inhibitors targeting the bc1 complex, dihydroorotate dehydrogenase and F1Fo ATP synthase complex. Furthermore, knockdown of the mitoribosome blocked the pyrimidine biosynthesis pathway and reduced the pool of pyrimidine nucleotides. Together, our data suggest that disruption of the P. falciparum mitoribosome compromises the metabolic capability of the mitochondrion, rendering the parasite hypersensitive to a panel of inhibitors targeting mitochondrial functions.

biochemistry↗

bioRxiv2019

Refining the transcriptome of the human malaria parasite Plasmodium falciparum using amplification-free RNA-seq

Plasmodium parasites undergo several major developmental transitions during their complex lifecycle, which are enabled by precisely ordered gene expression programs. Transcriptomes from the 48-hour blood stages of the major human malaria parasite Plasmodium falciparum have been described using cDNA microarrays and RNA-seq, but these assays have not always performed well within non-coding regions, where the AT-content is often 90-95%. We developed a directional, amplification-free RNA-seq protocol (DAFT-seq) to reduce bias against AT-rich cDNA, which we have applied to three strains of P. falciparum (3D7, HB3 and IT). While strain-specific differences were detected, overall there is strong conservation between the transcriptional profiles. For the 3D7 reference strain, transcription was detected from 89% of the genome, with over 75% of the genome transcribed into mRNAs. These datasets allowed us to refine the 5 and 3 untranslated regions (UTRs), which can be variable, long (>1,000 nt), and often overlap those of adjacent transcripts. We also find that transcription from bidirectional promoters frequently results in non-coding, antisense transcripts. By capturing the 5 ends of mRNAs, we reveal both constant and dynamic use of transcriptional start sites across the intraerythrocytic developmental cycle resulting in an updated view of the P. falciparum transcriptome.

genomics↗

bioRxiv2019

Hierarchical transcriptional control regulates Plasmodium falciparum sexual differentiation

Malaria pathogenesis relies on sexual gametocyte forms of the malaria parasite to be transmitted between the infected human and the mosquito host but the molecular mechanisms controlling gametocytogenesis remains poorly understood. Here we provide a high-resolution transcriptome of Plasmodium falciparum as it commits to and develops through gametocytogenesis. The gametocyte-associated transcriptome is significantly different from that of the asexual parasites, with dynamic gene expression shifts characterizing early, intermediate and late-stage gametocyte development and results in differential timing for sex-specific transcripts. The striking transcriptional dynamics suggest strict transcriptional control during gametocytogenesis in P. falciparum, which we propose is mediated by putative regulators including epigenetic mechanisms (driving active repression of proliferation-associated processes) and a cascade-like expression of ApiAP2 transcription factors. The gametocyte transcriptome serves as the blueprint for sexual differentiation and will be a rich resource for future functional studies on this critical stage of Plasmodium development, as the intraerythrocytic transcriptome has been for our understanding of the asexual cycle.

biochemistry↗

bioRxiv2019

Regulation of sexual differentiation is linked to invasion in malaria parasites

In the malaria parasite Plasmodium falciparum, the switch from asexual multiplication to sexual differentiation into gametocytes is essential for transmission to mosquitos. One of the key determinants of sexual commitment is the transcription factor PfAP2-G, which has been proposed to orchestrate this crucial cell fate decision by driving expression of gametocyte genes. We show conclusively that PfAP2-G is a transcriptional activator of gametocyte genes and identify the earliest known markers expressed during commitment. Remarkably, we also find that in sexually committed cells, PfAP2-G is associated with the promoters of genes important for red blood cell invasion and activates them through its interactions with a second transcription factor. We thus demonstrate an intriguing transcriptional link between the apparently opposing processes of red blood cell invasion and gametocytogenesis that is coordinated by the master regulator PfAP2-G. This finding has important implications for the development of new anti-malarial drugs that block the invasion of red blood cells by sexually committed cells, thereby preventing parasite transmission.

microbiology↗

bioRxiv2018

Inducing controlled cell cycle arrest and re-entry during asexual proliferation of Plasmodium falciparum malaria parasites.

The life cycle of the malaria parasite Plasmodium falciparum is tightly regulated, oscillating between stages of intense proliferation and quiescence. Cyclic 48-hour asexual replication of Plasmodium is markedly different from cell division in higher eukaryotes, and mechanistically poorly understood. Here, we report tight synchronisation of malaria parasites during the early phases of the cell cycle by exposure to DL--difluoromethylornithine (DFMO), which results in the depletion of polyamines. This induces an inescapable cell cycle arrest in G1 (~15 hours post-invasion) by blocking G1/S transition. Cell cycle-arrested parasites enter a quiescent G0-like state but, upon addition of exogenous polyamines, re-initiate their cell cycle in a coordinated fashion. This ability to halt malaria parasites at a specific point in their cell cycle, and to subsequently trigger re-entry into the cell cycle, provides a valuable framework to investigate cell cycle regulation in these parasites. We therefore used gene expression analyses to show that re-entry into the cell cycle involves expression of Ca2+-sensitive (cdpk4 and pk2) and mitotic kinases (nima and ark2), with deregulation of the pre-replicative complex associated with expression of pk2. Changes in gene expression could be driven through transcription factors MYB1 and two ApiAP2 family members. This new approach to parasite synchronisation therefore expands our currently limited toolkit to investigate cell cycle regulation in malaria parasites.

cell biology↗

bioRxiv2018

Real-time in vivo Global Transcriptional Dynamics During Plasmodium falciparum Blood-stage Development

Genome-wide analysis of transcription in the human malaria parasite Plasmodium falciparum has revealed robust variation in steady-state mRNA abundance throughout the 48-hour intraerythrocytic developmental cycle (IDC) suggesting that this process is highly dynamic and tightly regulated. However, the precise timing of mRNA transcription and decay remains poorly understood due to the utilization of methods that only measure total RNA and cannot differentiate between newly transcribed, decaying and stable cellular RNAs. Here we utilize rapid 4-thiouracil (4-TU) incorporation via pyrimidine salvage to specifically label, capture and quantify newly-synthesized P. falciparum RNA transcripts at every hour throughout the IDC following erythrocyte invasion. This high resolution global analysis of the transcriptome captures the timing and rate of transcription for each newly synthesized mRNA in vivo, revealing active transcription throughout all stages of the IDC. To determine the fraction of active transcription and/or transcript stabilization contributing to the total mRNA abundance at each timepoint we have generated a statistical model to fit the data for each gene which reveals varying degrees of transcription and stabilization for each mRNA corresponding to developmental transitions and independent of abundance profile. Finally, our results provide new insight into co-regulation of mRNAs throughout the IDC through regulatory DNA sequence motifs associated with these processes, thereby expanding our understanding of P. falciparum mRNA dynamics.

microbiology↗

bioRxiv2018

Antimalarial pantothenamide metabolites target acetyl-CoA synthesis in Plasmodium falciparum

Malaria eradication is critically dependent on novel drugs that target resistant Plasmodium parasites and block transmission of the disease. Here we report the discovery of potent pantothenamide bioisosteres that are active against blood-stage P. falciparum and also block onward mosquito transmission. These compounds are resistant to degradation by serum pantetheinases, show favorable pharmacokinetic properties and clear parasites in a humanized rodent infection model. Metabolomics revealed that CoA biosynthetic enzymes convert pantothenamides into drug-conjugates that interfere with parasite acetyl-CoA anabolism. In vitro generated resistant parasites showed mutations in acetyl-CoA synthetase and acyl-CoA synthetase 11, confirming the key roles of these enzymes in the sensitivity to pantothenamides. These new pantothenamides provide a promising class of antimalarial drugs with a unique mode of action.\n\nOne sentence summaryPantothenamides form antimetabolites that interfere with acetyl-CoA metabolism in the human malaria parasite Plasmodium falciparum

pharmacology and toxicology↗

bioRxiv2017

A Specific Non-Bisphosphonate Inhibitor Of The Bifunctional Farnesyl/Geranylgeranyl Diphosphate Synthase In Malaria Parasites

Isoprenoid biosynthesis is essential for Plasmodium falciparum (malaria) parasites and contains multiple validated antimalarial drug targets, including a bifunctional farnesyl and geranylgeranyl diphosphate synthase (FPPS/GGPPS). We identified MMV019313 as an inhibitor of PfFPPS/GGPPS. Though PfFPPS/GGPPS is also inhibited by a class of bisphosphonate drugs, MMV019313 has significant advantages for antimalarial drug development. MMV019313 has superior physicochemical properties compared to charged bisphosphonates that have poor bioavailability and strong bone affinity. We also show that it is highly selective for PfFPPS/GGPPS and showed no activity against human FPPS or GGPPS. Inhibition of PfFPPS/GGPPS by MMV019313, but not bisphosphonates, was disrupted in an S228T variant, demonstrating that MMV019313 and bisphosphonates have distinct modes-of-inhibition against PfFPPS/GGPPS. Altogether MMV019313 is the first specific, non-bisphosphonate inhibitor of PfFPPS/GGPPS. Our findings uncover a new small molecule binding site in this important antimalarial drug target and provide a promising starting point for development of Plasmodium-specific FPPS/GGPPS inhibitors.

microbiology↗