Biology subjects

Vembar, S. S.

Publications and source records attributed to Vembar, S. S..

6 recordsLinked to original sources

bioRxiv2026

Genomic 8-oxoguanine is associated with transcriptionally active chromatin and elevated gene expression in Plasmodium falciparum

Epigenetic regulation is central to the developmental progression and pathogenicity of the unicellular eukaryotic parasite Plasmodium falciparum; yet, the contribution of DNA base modifications remains poorly understood. One such modification, 8-oxoguanine (8-oxoG), which was initially identified as an oxidative lesion and a marker of DNA damage, has since emerged as a transcriptional regulator in advanced eukaryotes. Given that P. falciparum encounters a highly oxidative environment in human blood, we investigated the potential gene regulatory role of 8-oxoG during its intra-erythrocytic developmental cycle (IDC). Using immunodetection assays, we first confirmed the presence of 8-oxoG in P. falciparum genomic DNA and observed a gradual increase in 8-oxoG abundance from ring to schizont stages. We then optimized oxidative DNA immunoprecipitation sequencing (OxiDIP-seq) for the highly AT-rich parasite genome and generated genome-wide 8-oxoG profiles across four IDC timepoints, which revealed reproducible enrichment of 8-oxoG at discrete genomic loci, with more than 50% of the peaks stable across developmental stages. Notably, 8-oxoG accumulated at putative G-quadruplex-forming sequences in the parasite genome and preferentially localized within exonic regions of protein-coding genes, exhibiting a marked enrichment near STOP codons and within 3' untranslated regions. This in turn correlated with significantly higher steady-state transcript levels of 8-oxoG-marked genes, with stage-specific changes in 8-oxoG enrichment closely matching transcriptional activity. Furthermore, 8-oxoG-marked loci were preferentially associated with active and poised histone post-translational modifications, while showing no evidence of altered nucleosome occupancy. Collectively, these findings demonstrate that 8-oxoG is a widespread and non-random DNA modification in P. falciparum and suggest that it may function as an epigenetic mark associated with transcriptionally permissive chromatin and gene activation during parasite blood-stage development.

genomics↗

bioRxiv2026

DNA 6mA marks transcriptionally active chromatin in malaria parasites

DNA N6-methyladenine (6mA) has emerged as a significant epigenetic modification across a broad range of eukaryotes, from unicellular protists to metazoa. However, its role in unicellular eukaryotic parasites with highly AT-rich genomes, such as malaria-causing Plasmodium falciparum, remains unclear. Using mass spectrometry, South-western blotting, and Single Molecule Real-Time sequencing (Pacific Biosciences) across four stages of P. falciparum intra-erythrocytic development (IED), we confirmed that 0.02-0.04% of genomic adenines are modified to 6mA, with over 60% of the sites being stably maintained during the IED cycle. Notably, 6mA is enriched at transcription start sites, with genes bearing 6mA marks within their 5 and 3 untranslated regions exhibiting significantly elevated steady-state transcript levels. Consistent with this, 6mA loci show a strong positive correlation with activating histone post-translational modifications, while showing no significant association with repressive histone marks. Furthermore, in contrast to unicellular ciliates such as Oxytricha and Tetrahymena - organisms that share ancestry with Plasmodium - 6mA-marked genomic regions do not occlude nucleosomes. Lastly, we identified a putative 6mA methyltransferase belonging to the METTL4 family in P. falciparum, PfN6AMT encoded by the PF3D7_1303100 gene, and demonstrate that recombinant PfN6AMT exhibits robust methyltransferase activity in vitro, with mutation of its active site residues abolishing catalytic activity. Collectively, our findings demonstrate that 6mA is a low-abundance, yet reproducible, feature of the P. falciparum epigenome that is associated with transcriptionally active chromatin, and that the molecular mechanisms governing DNA adenine methylation may have undergone substantial evolutionary divergence, even among closely related eukaryotic lineages.

genomics↗

bioRxiv2024

Sequence, Structural and Functional Diversity of the Ubiquitous DNA/RNA-Binding Alba Domain

The DNA/RNA-binding Alba domain is prevalent across all kingdoms of life. Discovered in archaea more than 40 years ago, this protein domain appears to have evolved from RNA- to DNA- binding, with a concomitant expansion in the range of cellular processes that it regulates. Indeed, Alba domain-containing proteins continue to exhibit functional ingenuity, with their most recent association being R-loop regulation in plants: they accomplish this by binding to DNA:RNA hybrid structures as heterodimers. To further explore Alba diversity and evolutionary relatedness, which could in turn lead to new functional revelations, we employed iterative searches in PSI-BLAST to identify 15161 true Alba domain-containing proteins from the NCBI non-redundant protein database. Next, by building sequence similarity networks (SSNs), we identified 13 distinct clusters representing various Alba subgroups. These included the classic eukaryotic Rpp20/Pop7 and Rpp25/Pop6 proteins as well as novel fungal Alba proteins and Plasmodium-specific Albas. Conservation analysis indicated that, despite overall low primary sequence similarity between the SSN clusters, the homo- or hetero-dimer interface is highly conserved. Furthermore, the presence of the classic Alba fold was confirmed in representative sequences from each cluster by comparison of their Hidden Markov Model (HMM) profiles and ab initio three-dimensional structures. Notably, Alba domains from lower, unicellular eukaryotes and fungal Albas exhibit structural deviations towards their C-terminal end. Finally, phylogenetic analysis, while supporting SSN clustering, revealed the evolutionary branchpoint at which the eukaryotic Rpp20- and Rpp25-like clades emerged from archaeal Albas, and the subsequent taxonomic lineage-based divergence within each clade. Taken together, this comprehensive analysis enhances our understanding of the evolutionary history of Alba domain-containing proteins across diverse organisms, their sequence and structural conservation, and functional implications for genome and RNA biology.

bioinformatics↗

bioRxiv2021

Localization of the putative recombinase Pf-int to the apicoplast of Plasmodium falciparum

Diseases caused by apicomplexan parasites, such as malaria and toxoplasmosis cause [~]200 million (worldwide) and 1 million (Europe) infections, respectively, every year. Apicomplexa possess a non-photosynthetic organelle homologous to the plant chloroplast, the so-called apicoplast, that is essential for their growth and survival. This study focused on the Int recombinase, the first protein discovered in Plasmodium spp. with the features of a site-specific recombinase, and which has an apicoplast targeting leader sequence at its amino-terminus. Int is conserved amongst several apicomplexan parasites. In the human toxoplasmosis parasite, Toxoplasma, Int localizes to the apicoplast and Pf-Int, the P. falciparum member, belongs to the group of non-mutable essential genes in P. falciparum. A conserved protein that has been shown to be essential at least in one species and that localizes to an essential organelle may become a novel drug target. Therefore, the aim of this study was to confirm the sub-cellular localization of Int in the human malaria parasite P. falciparum. Using western blot analysis and immunofluorescence microscopy of P. falciparum asexual blood stages, we observed that Int partially co-localized with the apicoplast (to discrete foci adjacent to the nucleus).

biochemistry↗

bioRxiv2020

Whole Genome Sequences of Aedes aegypti (Linn.) Field Isolates from Southern India

Aedes spp. mosquitoes are a major health concern as they transmit several viral pathogens resulting in millions of deaths annually around the world. This is compounded by the emergence of insecticide-resistant strains and global warming, which could expose more than half of the world's population to Aedes-borne diseases in the future. Therefore, a comprehensive understanding of vector biology and the genomic basis of phenotypes such as insecticide resistance in natural populations are of paramount importance. Here, we sequenced the genome of Aedes aegypti mosquitos sampled from dengue-endemic areas and investigated the genetic variations between the previously reported laboratory-reared strain and our field isolates. The mosquito genomic DNA was used for paired-end sequencing using the Illumina platform. The reads were used for template-based assembly and mapped to the Aedes aegypti reference genome. Stringent parameters and multiple variant calling methods were used to identify unique single nucleotide variants (SNVs) and insertions-deletions (indels) and mapped to the Aedes chromosomes to create a draft consensus genome. Gene Ontology analyses was performed on the variant-enriched genes while two gene families involved in insecticide resistance were used for comparative sequence and phylogenetic analyses. Comparative sequence variant analyses showed that the majority of the high-quality variants in our samples mapped to non-coding regions of the genome, while gene ontology analyses of genic variants revealed enrichment of terms relevant to drug binding and insecticide resistance. Importantly, one mutation implicated in pyrethroid resistance was found in one Aedes sample. This is the first report of genome sequences of A. aegypti field isolates from India which reveals variants specific to the wild population. This is a useful resource which will facilitate development of robust integrated vector control strategies for management of Aedes-borne diseases through genetic manipulation of local mosquito populations.

genomics↗

bioRxiv2019

PfGCN5, a global regulator of stress responsive genes, modulates artemisinin resistance in Plasmodium falciparum

Plasmodium falciparum has evolved resistance to almost all front-line drugs including artemisinins, which threatens malaria control and elimination strategies. Oxidative stress and protein damage responses have emerged as key players in the generation of artemisinin resistance. In this study, we show that PfGCN5, a histone acetyltransferase, binds to the stress responsive and multi-variant family genes in poised state and regulates their expression under stress conditions. We have also provided biochemical and cellular evidences that PfGCN5 regulates stress responsive genes by acetylation of PfAlba3. Furthermore, we show that upon artemisinin exposure, genome-wide binding sites for PfGCN5 are increased and it is directly associated with the genes implicated in artemisinin resistance generation like BiP and TRiC chaperone. Moreover, inhibition of PfGCN5 in artemisinin resistant parasites, Kelch13 mutant, K13I543T and K13C580Y (RSA[~] 25% and 6%, respectively) reverses the sensitivity of the parasites to artemisinin treatment indicating its role in drug resistance generation. Together, these findings elucidate the role of PfGCN5 as a global chromatin regulator of stress-responses with potential role in modulating artemisinin drug resistance, and identify PfGCN5 as an important target against artemisinin resistant parasites.\n\nAuthor SummaryMalaria parasites are constantly adapting to the drugs we used to eliminate them. Thus, when we use the drugs to kill parasites; with time, we select the parasites with the favourable genetic changes. Parasites develop various strategies to overcome exposure to the drugs by exhibiting the stress responses. The changes specific to the drug adapted parasites can be used to understand the mechanism of drug resistance generation. In this study, we have identified PfGCN5 as a global transcriptional regulator of stress responses in Plasmodium falciparum. Inhibition of PfGCN5 reverses the sensitivity of the parasites to the artemisinin drug and identify PfGCN5 as an important target against artemisinin resistant parasites.

cell biology↗