Biology subjects

Tripathi, A. K.

Publications and source records attributed to Tripathi, A. K..

4 recordsLinked to original sources

bioRxiv2023

Mapping the genomic landscape of multidrug resistance in Plasmodium falciparum and its impact on parasite fitness

Drug-resistant Plasmodium falciparum parasites have swept across Southeast Asia and now threaten Africa. By implementing a P. falciparum genetic cross using humanized mice, we report the identification of key determinants of resistance to artemisinin (ART) and piperaquine (PPQ) in the dominant Asian KEL1/PLA1 lineage. We mapped k13 as the central mediator of ART resistance and identified secondary markers. Applying bulk segregant analysis, quantitative trait loci mapping and gene editing, our data reveal an epistatic interaction between mutant PfCRT and multicopy plasmepsins 2/3 in mediating high-grade PPQ resistance. Susceptibility and parasite fitness assays implicate PPQ as a driver of selection for KEL1/PLA1 parasites. Mutant PfCRT enhanced susceptibility to lumefantrine, the first-line partner drug in Africa, highlighting a potential benefit of opposing selective pressures with this drug and PPQ. We also identified that the ABCI3 transporter can operate in concert with PfCRT and plasmepsins 2/3 in mediating multigenic resistance to antimalarial agents.

genomics↗

bioRxiv2023

Transmissibility of clinically relevant atovaquone-resistant Plasmodium falciparum by anopheline mosquitoes

Rising numbers of malaria cases and deaths underscore the need for new interventions. Long-acting injectable medications, such as those now in use for HIV prophylaxis, offer the prospect of a malaria "chemical vaccine", combining the efficacy of a drug (like atovaquone) with the durability of a biological vaccine. Of concern, however, is the possible selection and transmission of drug-resistant parasites. We addressed this question by generating clinically relevant, highly atovaquone-resistant, Plasmodium falciparum mutants competent to infect mosquitoes. Isogenic paired strains, that differ only by a single Y268S mutation in cytochrome b, were evaluated in parallel in southeast Asian (Anopheles stephensi) or African (Anopheles gambiae) mosquitoes, and thence in humanized mice. Fitness costs of the mutation were evident along the lifecycle, in asexual parasite growth in vitro and in a progressive loss of parasites in the mosquito. In numerous independent experiments, microscopic exam of salivary glands from hundreds of mosquitoes failed to detect even one Y268S sporozoite, a defect not rescued by coinfection with wild type parasites. Furthermore, despite uniformly successful transmission of wild type parasites from An. stephensi to FRG NOD huHep mice bearing human hepatocytes and erythrocytes, multiple attempts with Y268S-fed mosquitoes failed: there was no evidence of parasites in mouse tissues by microscopy, in vitro culture, or PCR. These studies confirm a severe-to-lethal fitness cost of clinically relevant atovaquone-resistant P. falciparum in the mosquito, and they significantly lessen the likelihood of their transmission in the field. SignificanceNew tools are needed to protect individuals from malaria and to control malaria in the field. Atovaquone plus proguanil is a commonly used and well-tolerated medicine to prevent malaria. No drug resistance has been reported from its prophylactic use, but tablets must be taken daily. Giving atovaquone as a single injection may provide much longer-lasting protection, against both falciparum and vivax malaria, but there is concern this may create drug resistance. In this study we showed that clinically relevant atovaquone-resistant malaria parasites survive poorly, if at all, in mosquitoes, and that mosquitoes do not transmit drug-resistant parasites to humanized mice. These findings lessen the likelihood that an atovaquone "chemical vaccine" would lead to the spread of atovaquone resistance.

microbiology↗

bioRxiv2022

Deciphering the non-coding code of pathogenicity and sexual differentiation in the human malaria parasite.

The complex life cycle of Plasmodium falciparum requires coordinated gene expression regulation to allow host cell invasion, transmission, and immune evasion. However, this cascade of transcripts is unlikely to be regulated by the limited number of identified parasite-specific transcription factors. Increasing evidence now suggests a major role for epigenetic mechanisms in gene expression in the parasite. In eukaryotes, many lncRNAs have been identified and shown to be pivotal regulators of genome structure and gene expression. To investigate the regulatory roles of lncRNAs in P. falciparum we explored the intergenic lncRNA distribution in nuclear and cytoplasmic subcellular locations. Using nascent RNA expression profiles, we identified a total of 1,768 lncRNAs, of which 58% were identified as novel lncRNAs in P. falciparum. The subcellular localization and stage-specific expression of several putative lncRNAs were validated using RNA fluorescence in situ hybridization (RNA-FISH). Additionally, the genome-wide occupancy of several candidate nuclear lncRNAs was explored using Chromatin Isolation by RNA Purification (ChIRP). ChIRP-seq of candidate lncRNAs revealed that lncRNA occupancy sites within the parasite genome are focal and sequence-specific with a particular enrichment for several parasite-specific gene families, including those involved in pathogenesis, erythrocyte remodeling, and regulation of sexual differentiation. We further validated the function of one specific lncRNA (lncRNA-ch14) using the CRISPR-Cas9 genome editing tool. Genomic and phenotypic analysis of the {bigtriangleup}lncRNA-ch14 line demonstrated the importance of this lncRNA in sexual differentiation and sexual reproduction. Our findings bring a new level of insight into the role of lncRNAs in pathogenicity, gene regulation and sexual differentiation. These findings also open new avenues for targeted approaches towards therapeutic strategies against the deadly malaria parasite.

microbiology↗

bioRxiv2021

Can active hydrodynamic fluctuations affect barrier crossing during enzymatic catalysis?

The cellular milieu is teeming with biochemical nano-machines whose activity is a strong source of correlated non-thermal fluctuations termed active noise. Essential elements of this circuitry are enzymes, catalysts that speed up the rate of metabolic reactions by orders of magnitude, thereby making life possible. Here, we examine the possibility that active noise in the cell, or in vitro, affects enzymatic catalytic rate by accelerating or decelerating the crossing rate of energy barriers during the reaction. Considering hydrodynamic perturbations induced by biochemical activity as a source of active noise, we evaluate their impact on the enzymatic cycle using a combination of analytic and numerical methods. Our estimates show that the fast component of the active noise spectrum enhances the rate of enzymes, while reactions remain practically unaffected by the slow noise spectrum. Revisiting the physics of barrier crossing under the influence of active hydrodynamic fluctuations suggests that the biochemical activity of macromolecules such as enzymes is coupled to active noise. Thus, we propose that enzymatic catalysis is a collective, many-body process in which enzymes may affect each others activity via long-range hydrodynamic interaction, with potential impact on biochemical networks in living and artificial systems alike.

biophysics↗