Two orthogonal systems for inducible transcriptional repression in the malaria parasite Plasmodium falciparum using bacterial repressor proteins are created, improving on previous attempts at transcriptional regulation by two orders of magnitude and outperforming gold standard translational/post-transcriptional regulation systems.
Abstract
Malaria is responsible for over half a million deaths each year. However, our understanding of malaria parasite biology is hampered by a lack of molecular tools, particularly at the level of transcriptional control. In light of this, we have created two orthogonal systems for inducible transcriptional repression in the malaria parasite Plasmodium falciparum using bacterial repressor proteins. We achieve 200- to 800-fold repression of expression, improving on previous attempts at transcriptional regulation by two orders of magnitude and outperforming gold standard translational/post-transcriptional regulation systems. We developed automated DNA design software to apply this tool to conditional regulation of native gene expression, validating essentiality and chemogenetic interactions with both two parasite lipid kinases and PfKelch13, which is associated with artemisinin resistance. These tools can advance our understanding and engineering of malaria functional genomics, drug mechanisms, and gene regulation.
Background Malaria remains a global health challenge, with most fatal cases caused by
Plasmodium falciparum
in sub-Saharan Africa. A critical bottleneck in the
Plasmodium
life cycle is the transmission from human to mosquito, which requires a subset of blood stage parasites to switch from asexual to sexual replication in a process known as stage conversion. In
P. falciparum
, stage conversion is known to be environmentally sensitive, notably to limiting levels of the immunomodulatory lipid lysophosphatidylcholine (LysoPC). Methods Here, we use single cell RNA sequencing (scRNAseq) to deconvolve the transcriptional signature of parasites upon limiting LysoPC levels under controlled
in vitro
conditions. Results Our data validate previous findings from bulk RNA sequencing, demonstrating a general compensatory metabolic response and reduced nutrient transport triggered by reduced LysoPC levels. Further, we define an initial transcriptional signature of sexual commitment, consisting of 22 genes conserved across two genetically diverse parasite strains. This signature includes an entire genetic locus encoding three proteins putatively involved in host-parasite interactions. Interestingly, the proportion of parasites expressing the transcriptional signature of sexual commitment was about twofold higher than the sexual conversion rate experimentally measured in the subsequent gametocyte cycle. Conclusions Together with recent data demonstrating that sexual commitment and conversion rates as measured by fluorescent reporter lines match closely, these data suggest that sexually committed schizonts may be in a transcriptionally bipotential state and sexual development becomes irreversible only at the post transcriptional level. Our study provides a rationale for systematic functional dissection of this elusive yet essential phase in the parasite cycle.
Lauren Galloway, Fiona Achcar, Barbara H. Stokes et al.· Wellcome Open Research· 0 citations
Plasmodium falciparum, the primary cause of human malaria, relies on tightly coordinated gene-expression programs to adapt to host-derived stress despite possessing a limited repertoire of canonical transcription factors. Antisense long noncoding RNAs have emerged as important regulators of parasite biology, including virulence gene regulation and sexual commitment; however, their prevalence, origin, and broader functional significance remain poorly understood. Here, we demonstrate that antisense transcription is a widespread, reproducible, and regulated feature of the P. falciparum transcriptome rather than a byproduct of pervasive euchromatic transcription. Environmental stress, including febrile temperature exposure and artemisinin treatment, extensively remodelled antisense transcription, particularly at loci associated with virulence and stress adaptation, promoting widespread sense-antisense RNA duplex formation. Functional analyses of two stress-responsive chromatin regulators, PfGCN5 and PfHDAC1, identified as antisense-expressing loci, revealed that increased antisense expression elevated steady-state mRNA abundance while reducing cognate protein levels. Mechanistically, sense-antisense RNA duplex formation stabilized complementary transcripts but suppressed translation. Integrated transcriptomic, RNA-RNA duplex profiling, ribosome sequencing, and proteomic analyses further showed that duplex-enriched transcripts exhibit reduced ribosome occupancy and reduced protein abundance, accompanied by localized antisense enrichment near transcription end sites and altered ribosome distribution consistent with impaired translational engagement. Collectively, our findings identify an antisense RNA-ribosome regulatory axis that couples RNA duplex formation to adaptive translational control, providing a previously unrecognized mechanism underlying stress adaptation and post-transcriptional gene regulation in P. falciparum.
R. Malhotra, Kushankur Pandit, Pawan Malhotra et al.· RNA: A publication of the RN...· 0 citations
Epigenetic pathways have many important roles controlling virulence in human malaria parasites. Histone acetylation and methylation have been closely studied in this context but the novel epigenetic mark of lactylation has not yet been examined. Here, for the first time, we profiled lactyl-histone marks across the P. falciparum genome and found them strongly enriched at virulence genes, including genes involved in cytoadhesion and other host-cell remodelling functions. Many genes were dynamically and inducibly lactylated across the cell cycle. Thus, P. falciparum could use histone lactylation to control its virulence pathways in response to the prevailing metabolic environment in its host. We extended our chromatin profiling to parasites isolated directly from human patients, showing that here too, virulence gene families were strongly lactylated. This represents the first comprehensive profiling of P. falciparum chromatin from parasites in sub-millilitre blood samples, opening up exciting new avenues to study parasite chromatin across human disease states.
I. Jabre, Nana Efua Andoh, Haddijatou Mbye et al.· bioRxiv· 0 citations
Malaria is one of the world’s most critical parasitic diseases; it is caused by Plasmodium species and spreads through Anopheles mosquitoes. The life cycle of Plasmodium species is complex and involves several stages in humans and mosquitoes, each involving distinct molecular interactions. Recent molecular biology research has suggested that host microRNAs (miRNAs) may serve as important regulators of host–parasite interactions during malaria infection. MicroRNAs, approximately 22 nucleotides long, are non-coding RNAs that control gene expression by binding to target mRNAs and either repressing translation or causing mRNA degradation. During infection, host miRNAs are actively regulated and can directly target parasite transcripts or influence host cellular pathways essential for parasite survival and disease development. The role of human miRNAs has not yet been fully explored in malaria. This report aims to integrate evidence from the literature, molecular mechanisms, and translational insights to summarize the current knowledge in miRNA research and to guide the diagnosis and treatment of malaria using miRNAs. Here, we focus on the mechanistic and cell-type-resolved dimension of host miRNA action in malaria: the extracellular vesicle-mediated transfer of AGO2–miRNA complexes between infected erythrocytes and recipient endothelial cells, the bidirectional host–parasite miRNA traffic that allows human miRNAs to repress parasite transcripts, and the tissue-specific miRNA programs that shape organ-specific pathology. We also delineate the principal knowledge gaps related to causality versus correlation, cell-of-origin attribution, and translational barriers to miRNA therapeutics that must be resolved before miRNAs can be deployed diagnostically or therapeutically in malaria.
N. Metwally, Maria del Pilar Martínez Tauler, Hanifeh Torabi et al.· Frontiers in Cellular and In...· 0 citations
Temperature is a key determinant of malaria transmission, influencing both parasite development and mosquito physiology, yet the underlying mechanisms remain poorly understood. Here, we examined how temperature and time modulate gene expression in Anopheles stephensi infected with Plasmodium falciparum. Using RNA-sequencing over 1-19 days post-blood meal and three temperature regimes (20, 24, and 28°C with diurnal fluctuations of 9°C), we characterize transcriptome responses to infection with P. falciparum at the site of infection in the midgut, and systemically, in the carcasses. Oocyst prevalence and density declined over the thermal gradient, albeit with distinct, non-linear temporal dynamics in parasite development rates. Although infection contributed minimally to global variation in gene expression relative to temperature and time, infection-associated genes in the midgut showed coordinated transcriptional responses enriched in canonical Plasmodium associated extracellular, proteolytic, immune, and metabolic functions; notably, decline in oocyst infections in the midguts over the thermal gradient was reflected in reduced expression of immune genes known to regulate P. falciparum. Network analysis demonstrated that these genes participate in a significantly interconnected protein–protein interaction network, within which a small number of high betweenness centrality proteins act as bottlenecks linking immune, metabolic, reproductive and behavioral processes. Our results suggest responses to infection may be mediated through coordinated physiological networks rather than large-scale transcriptional changes. Our findings also indicate that differences in thermal conditions may be an important factor when comparing mechanisms of vector– parasite interactions between Plasmodium species. Together, our results highlight the importance of integrating thermal context into mechanistic studies of vector–parasite interactions.
Ashutosh K. Pathak, Shannon Quek, Ritu Sharma et al.· bioRxiv· 0 citations
Plasmodium falciparum, the causative agent of malaria, undergoes a critical cell fate switch during the erythrocytic stage, when a subset of parasites exits asexual replication and differentiates into gametocytes-the forms required for transmission. This conversion is controlled by the transcription factor AP2-G, whose expression is normally repressed by H3K9me3 marked heterochromatin. The mechanism by which this silenced state is abolished has remained unknown. Here we identify a cis-regulatory element, URE-G, that is required for high levels of H3K9 methylation at the ap2-g locus. Deletion of URE-G decreases this modification, resulting in AP2-G activation and increased gametocyte formation, without altering global heterochromatin structure. These findings suggest a sequence-dependent mechanism for regulating heterochromatinized genes and provide insights into cell fate determination in malaria parasites.
Mai Nakashima, Shiroh Iwanaga, Toshiyuki Mori· Scientific Reports· 0 citations
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