Biology subjects

Ramaprasad, A.

Publications and source records attributed to Ramaprasad, A..

12 recordsLinked to original sources

bioRxiv2025

Molecular and functional dissection of the AP2-I/BDP1 transcriptional complex of the malaria parasite reveals a role beyond the red blood cell stages

The transcription factor AP2-I and the bromodomain protein BDP1 of the malaria parasite bind upstream of several genes implicated in red blood cell invasion. Here, we demonstrate that they form a complex and that AP2-I recruits BDP1 to the target genes. The ACDC domain of AP2-I interacts with a BDP1 region called BAAS, located between the ankyrin and the bromodomain. The ankyrin, in turn, binds to a conserved AP2-I specific region called ASAIR. Three of the interacting domains are parasite-specific, allowing inhibition without affecting the host. We further show that AP2-I is essential for asexual blood stage development, playing multiple roles, and that AP2-I and BDP1 function beyond invasion, binding to the promoters of the gametocytogenesis regulators gdv1 and ap2-g, suggesting a role for the AP2-I/BDP1 complex in parasite sexual commitment. Overall, our study highlights the essential and unique nature of the AP2-I/BDP1 complex and its potential as a novel antimalarial drug target. Author summaryThe transcription factor AP2-I and the bromodomain protein BDP1 of the malaria parasite have previously been shown to be implicated in red blood cell invasion by the human malaria parasite. Here we show that they form a complex and identify the interacting domains on each protein. Three of these are parasite-specific, allowing inhibition without affecting the human host. We also show that AP2-I is essential for parasite survival in the blood and that the protein is implicated in other processes beyond red blood cell invasion. Namely, AP2-I, but also BDP1, bind to the promoters of the gametocytogenesis regulators gdv1 and ap2-g, suggesting a role for the AP2-I/BDP1 complex in parasite sexual commitment, without which the parasite cannot be transmitted.

microbiology↗

bioRxiv2024

A scaleable inducible knockout systemfor studying essential gene function inthe malaria parasite

The malaria parasite needs nearly half of its genes to propagate normally within red blood cells. Inducible ways to interfere with gene expression like the DiCre-lox system is necessary to study the function of these essential genes. However, the existing DiCre-lox strategy is not well-suited to be deployed at scale to study several genes simultaneously. To overcome this, we have developed SHIFTiKO (frameshift-based trackable inducible knockout), a novel scaleable strategy that uses short, easy-to-construct, barcoded repair templates to insert loxP sites around short regions in the target genes. Induced DiCre-mediated excision of the flanked region causes a frameshift mutation resulting in genetic ablation of gene function. Dual DNA barcodes inserted into each mutant enables verification of successful modification and induced excision at each locus and collective phenotyping of the mutants, not only across multiple replication cycles to assess growth fitness but also within a single cycle to identify the specific phenotypic impairment they exhibit. As a proof of concept, we have applied SHIFTiKO to screen the functions of malarial rhomboid proteases, successfully identifying their blood stage-specific essentiality. SHIFTiKO, thus offers a powerful platform to conduct inducible phenotypic screens to study essential gene function at scale in the malaria parasite.

microbiology↗

bioRxiv2023

A malaria parasite phospholipase facilitates efficient asexual blood stage egress

Malaria parasite release (egress) from host red blood cells involves parasite-mediated membrane poration and rupture, thought to involve membrane-lytic effector molecules such as perforin-like proteins and/or phospholipases. With the aim of identifying these effectors, we disrupted the expression of two Plasmodium falciparum perforin-like proteins simultaneously and showed that they have no essential roles during blood stage egress. Proteomic profiling of parasite proteins discharged into the parasitophorous vacuole (PV) just prior to egress detected the presence in the PV of a lecithin:cholesterol acyltransferase (LCAT; PF3D7_0629300). Conditional ablation of LCAT resulted in abnormal egress and a reduced replication rate. Lipidomic profiles showed drastic changes in several phosphatidylserine and acylphosphatidylglycerol species during egress. We thus show that, in addition to its previously demonstrated role in liver stage merozoite egress, LCAT is required to facilitate efficient egress in asexual blood stage malaria parasites. Author SummaryMalaria kills over half a million people every year worldwide. It is caused by a single-celled parasite called Plasmodium falciparum that grows and multiplies within a bounding vacuole, inside red blood cells of the infected individuals. Following each round of multiplication, the infected cell is ruptured in a process known as egress to release a new generation of parasites. Egress is required for the disease to progress and is orchestrated by the parasite. The parasite sends out various molecules to puncture and destroy the membranes of the vacuole and the red blood cell. However, little is known about these molecules. In this work, we set out to identify these molecules by using genetic and proteomics approaches. We screened the molecules the parasite sends out during egress and identified a parasite enzyme called LCAT present in the vacuole. Our experiments found that mutant parasites that were unable to make LCAT clumped together and could not escape the infected cell properly. As a result, we saw a reduction in the rate at which these parasites spread through the red blood cells. Taken together, our findings suggest that P. falciparum needs LCAT to efficiently break out of red blood cells.

microbiology↗

bioRxiv2022

A choline-releasing glycerophosphodiesterase essential for phosphatidylcholine biosynthesis and blood stage development in the malaria parasite

The malaria parasite Plasmodium falciparum synthesizes significant amounts of phospholipids to meet the demands of replication within red blood cells. De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway is essential, requiring choline that is primarily sourced from host serum lysophosphatidylcholine (lysoPC). LysoPC also acts as an environmental sensor to regulate parasite sexual differentiation. Despite these critical roles for host lysoPC, the enzyme(s) involved in its breakdown to free choline for PC synthesis are unknown. Here we show that a parasite glycerophosphodiesterase (PfGDPD) is indispensable for blood stage parasite proliferation. Exogenous choline rescues growth of PfGDPD-null parasites, directly linking PfGDPD function to choline incorporation. Loss of PfGDPD reduces choline uptake from lysoPC, resulting in depletion of several PC species in the parasite, whilst purified PfGDPD releases choline from glycerophosphocholine in vitro. Our results identify PfGDPD as a choline-releasing glycerophosphodiesterase that mediates a critical step in PC biosynthesis and parasite survival.

microbiology↗

bioRxiv2021

Global analysis of putative phospholipases in the malaria parasite Plasmodium falciparum reveals critical factors for parasite proliferation

For its replication within red blood cells, the malaria parasite is highly dependent on correctly regulated lipid metabolism. Enzymes involved in lipid metabolic processes are therefore potential drug targets. We here provide a functional analysis of the 20 putative phospholipases that are expressed by asexual blood stages of Plasmodium falciparum. We reveal a high level of redundancy among members of this group, but using conditional mislocalization and gene disruption techniques we show that the phosphoinositide-specific phospholipase C (PF3D7_1013500) has a previously unrecognized essential role in intracellular parasite maturation. In addition, we demonstrate that the patatin-like phospholipase PF3D7_1358000 localizes to the mitochondrion. Parasites lacking this enzyme display a severe growth phenotype and defects in mitochondrial morphogenesis and function leading to hypersensitivity towards proguanil and inhibitors of the mitochondrial electron transport chain including atovaquone. This demonstrates that regulated mitochondrial lipid homeostasis is necessary for mitochondrial function and coordinated division during parasite multiplication.

microbiology↗

bioRxiv2021

Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome

Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal excitation. In exploring the basis of these N-glycosylation alterations, we discovered they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed with neuronal excitation were in close association with ER exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway, or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrites satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction and disease.

neuroscience↗

bioRxiv2020

The pangenome analysis of the soil-borne fungal phytopathogen Rhizoctonia solaniand development of a comprehensive web resource: RsolaniDB

Rhizoctonia solani is a collective group of genetically and pathologically diverse basidiomycetous fungus that damages economically important crops. Its isolates are classified into 13 Anastomosis Groups (AGs) and subgroups having distinctive morphology and host range. The genetic factors driving the unique features of R. solani pathology are not well characterized due to the limited availability of its annotated genomes. Therefore, we performed genome sequencing, assembly, annotation and functional analysis of 12 R. solani isolates covering 7 AGs and selected subgroups (AG1-IA, AG1-IB, AG1-IC, AG2-2IIIB, AG3-PT (isolates Rhs 1AP and the hypovirulent Rhs1A1), AG3-TB, AG4-HG-I (isolates Rs23 and R118-11), AG5, AG6, and AG8), in which six genomes are reported for the first time, wherein we discovered unique and shared secretomes, CAZymes, and effectors across the AGs. Using a pangenome comparative analysis of 12 R. solani isolates and 15 other basidiomycetes, we also elucidated the molecular factors potentially involved in determining the AG-specific host preference, and the attributes distinguishing them from other Basidiomycetes. Finally, we present the largest repertoire of R. solani genomes and their annotated components as a comprehensive database, viz. RsolaniDB, with tools for large-scale data mining, functional enrichment and sequence analysis not available with other state-of-the-art platforms, to assist mycologists in formulating new hypotheses.

genomics↗

bioRxiv2020

Plasmodium vinckei genomes provide insights into the pan-genome and evolution of rodent malaria parasites

BackgroundRodent malaria parasites (RMPs) serve as tractable tools to study malaria parasite biology and host-parasite-vector interactions. Plasmodium vinckei is the most geographically widespread of the four RMP species collected in sub-Saharan Central Africa. Several P. vinckei isolates are available but relatively less characterized than other RMPs, thus hindering their use in experimental studies. We have generated a comprehensive resource for P. vinckei comprising of high-quality reference genomes, genotypes, gene expression profiles and growth phenotypes for ten P. vinckei isolates. ResultsThe P. vinckei subspecies have diverged widely from their common ancestor and have undergone genomic structural variations. The subspecies from Katanga, P. v. vinckei, has a uniquely smaller genome, a reduced multigene family repertoire and is also amenable to genetic manipulation making it an ideal parasite for reverse genetics. Comparing P. vinckei genotypes reveals region-specific selection pressures particularly on genes involved in mosquito transmission. The erythrocyte membrane antigen 1 and fam-c families have expanded considerably among the lowland forest-dwelling P. vinckei parasites. Genetic crosses can be established in P. vinckei but are limited at present by low transmission success under the experimental conditions tested in this study. ConclusionsPlasmodium vinckei isolates display a large degree of phenotypic and genotypic diversity and could serve as a resource to study parasite virulence and immunogenicity. Inclusion of P. vinckei genomes provide new insights into the evolution of RMPs and their multigene families. Amenability to genetic crossing and genetic manipulation make them also suitable for classical and functional genetics to study Plasmodium biology.

genomics↗

bioRxiv2020

MRE11 is crucial for malaria transmission and its absence affects expression of interconnected networks of key genes essential for life

The Meiotic Recombination 11 protein (MRE11) plays a key role in DNA damage response and maintenance of genome stability. However, little is known about its function during development of the malaria parasite Plasmodium. Here, we present a functional, ultrastructural and transcriptomic analysis of Plasmodium MRE11 during its life-cycle in both mammalian and mosquito vector hosts. Genetic disruption of Plasmodium berghei mre11 (PbMRE11) results in significant retardation of oocyst development in the mosquito midgut associated with cytoplasmic and nuclear degeneration, along with concomitant ablation of sporogony and subsequent parasite transmission. Further, absence of PbMRE11 results in significant transcriptional downregulation of genes involved in key interconnected biological processes that are fundamental to all eukaryotic life including ribonucleoprotein biogenesis, spliceosome function and iron-sulphur cluster assembly. Overall, our study provides a comprehensive functional analysis of MRE11s role in Plasmodium development during the mosquito stages and offers a potential target for therapeutic intervention during malaria parasite transmission.

cell biology↗

bioRxiv2019

Disruption of the coordination between host circadian rhythms and malaria parasite development alters the duration of the intraerythrocytic cycle

Malaria parasites complete their intra-erythrocytic developmental cycle (IDC) in multiples of 24 hours (depending on the species), suggesting a circadian basis to the asexual cell cycle, but the mechanism controlling this periodicity is unknown. Combining in vivo and in vitro approaches using rodent and human malaria parasites, we reveal that: (i) 57% of Plasmodium chabaudi genes exhibit 24 h \"circadian\" periodicity in transcription; (ii) 58% of these genes lose transcriptional rhythmicity when the IDC is out-of-synchrony with host rhythms; (iii) 9% of Plasmodium falciparum genes show circadian transcription under free-running conditions; (iv) Serpentine receptor 10 (SR10) has a circadian transcription profile and disrupting it in rodent malaria parasites shortens the IDC by 2-3 hours; (v) Multiple processes including DNA replication and the ubiquitin and proteasome pathways are affected by loss of coordination with host rhythms and by disruption of SR10. Our results show that malaria parasites are at least partly responsible for scheduling their IDCs explaining the fitness benefits of coordination with host rhythms.

genomics↗

bioRxiv2018

A fast and cost-effective microsampling protocol incorporating reduced animal usage for time-series transcriptomics in rodent malaria parasites

The transcriptional regulation occurring in malaria parasites during the clinically important life stages within host erythrocytes can be studied in vivo with rodent malaria parasites propagated in mice. Time-series transcriptome profiling commonly involves the euthanasia of groups of mice at specific time points followed by the extraction of parasite RNA from whole blood samples. Current methodologies for parasite RNA extraction involve several steps and when multiple time points are profiled, these protocols are laborious, time consuming, and require the euthanisation of large cohorts of mice. We designed a simplified protocol for parasite RNA extraction from blood volumes as low as 20 microliters (microsamples), serially bled from mice via tail snips and directly lysed with TRIzol reagent. Gene expression data derived from microsampling using RNA-seq were closely matched to those derived from larger volumes of leucocyte-depleted and saponin-treated blood obtained from euthanized mice and also tightly correlated between biological replicates. Transcriptome profiling of microsamples taken at different time points during the intra-erythrocytic developmental cycle of the rodent malaria parasite Plasmodium vinckei revealed the transcriptional cascade commonly observed in malaria parasites. Microsampling is a quick, robust and cost-efficient approach to sample collection for in vivo time-series transcriptomic studies in rodent malaria parasites.

microbiology↗

bioRxiv2017

Timing of host feeding drives rhythms in parasite replication

Circadian rhythms enable organisms to synchronise the processes underpinning survival and reproduction to anticipate daily changes in the external environment. Recent work shows that daily (circadian) rhythms also enable parasites to maximise fitness in the context of ecological interactions with their hosts. Because parasite rhythms matter for their fitness, understanding how they are regulated could lead to innovative ways to reduce the severity and spread of diseases. Here, we examine how host circadian rhythms influence rhythms in the asexual replication of malaria parasites. Asexual replication is responsible for the severity of malaria and fuels transmission of the disease, yet, how parasite rhythms are driven remains a mystery. We perturbed feeding rhythms of hosts by 12 hours (i.e. diurnal feeding in nocturnal mice) to desynchronise the hosts peripheral oscillators from the central, light-entrained oscillator in the brain and their rhythmic outputs. We demonstrate that the rhythms of rodent malaria parasites in day-fed hosts become inverted relative to the rhythms of parasites in night-fed hosts. Our results reveal that the hosts peripheral rhythms (associated with the timing of feeding and metabolism), but not rhythms driven by the central, light-entrained circadian oscillator in the brain, determine the timing (phase) of parasite rhythms. Further investigation reveals that parasite rhythms correlate closely with blood glucose rhythms. In addition, we show that parasite rhythms resynchronise to the altered host feeding rhythms when food availability is shifted, which is not mediated through rhythms in the host immune system. Our observations suggest that parasites actively control their developmental rhythms. Finally, counter to expectation, the severity of disease symptoms expressed by hosts was not affected by desynchronisation of their central and peripheral rhythms. Our study at the intersection of disease ecology and chronobiology opens up a new arena for studying host-parasite-vector coevolution and has broad implications for applied bioscience.\n\nAuthor summaryHow cycles of asexual replication by malaria parasites are coordinated to occur in synchrony with the circadian rhythms of the host is a long-standing mystery. We reveal that rhythms associated with the time-of-day that hosts feed are responsible for the timing of rhythms in parasite development. Specifically, we altered host feeding time to phase-shift peripheral rhythms, whilst leaving rhythms driven by the central circadian oscillator in the brain unchanged. We found that parasite developmental rhythms remained synchronous but changed their phase, by 12 hours, to follow the timing of host feeding. Furthermore, our results suggest that parasites themselves schedule rhythms in their replication to coordinate with rhythms in glucose in the hosts blood, rather than have rhythms imposed upon them by, for example, host immune responses. Our findings reveal a novel relationship between hosts and parasites that if disrupted, could reduce both the severity and transmission of malaria infection.

microbiology↗