Biology subjects

Carrasquilla, M.

Publications and source records attributed to Carrasquilla, M..

8 recordsLinked to original sources

bioRxiv2025

Continental-scale genomic surveillance of Plasmodium falciparum malaria with rapid nanopore sequencing

In sub-Saharan Africa, continental-scale genomic surveillance of Plasmodium falciparum malaria is needed to track the spread of antimalarial drug resistance and diagnostic test evasion, as well as to monitor parasite evolutionary responses to vaccine rollout. Yet implementation of malaria genomic surveillance at a continental-scale is hindered by resource constraints, the vastness of the continent, and the lack of sequencing protocols suitable for most local laboratories. To address this, we developed an approach to enable a decentralized scale-up of P. falciparum genomic surveillance and established it in six African countries in one year, locally sequencing 1,065 samples. The approach includes a rapid ([~] 5 hours) and cost-efficient (<$25 USD/sample) nanopore sequencing protocol that provides surveillance of drug resistance-associated genes, hrp2/3 deletions, the vaccine target csp, and the polymorphic gene ama1. We coupled this to a bioinformatics dashboard that runs offline on a laptop and displays mapping and variant calling results in real-time. We demonstrate robust sequencing coverage across parasitemia levels and laboratories, accurate identification of antimalarial resistance markers and hrp2/3 deletions; and, with a novel variant caller, sensitive detection of mutations carried by minor clones. Our approach will accelerate genomic surveillance of P. falciparum malaria across sub-Saharan Africa at a time of urgent need.

genomics↗

bioRxiv2025

Seasonal transcriptional profiling of malaria parasites using a new single-cell atlas of a Malian Plasmodium falciparum isolate

Asymptomatic persistence is crucial for the survival of malaria parasites in seasonal transmission settings, where transmission halts during dry months, while mosquitos are scarce and new infections rare. How Plasmodium falciparum avoids causing host symptoms or being cleared is not fully understood. Parasites that persist through the dry season circulate longer within the [~]48h replication cycle compared to parasites isolated from clinical cases, where only early stages circulate. The mechanisms promoting increased circulation and avirulent infections bridging two wet seasons remain unclear. We generated a single-cell RNA-seq reference atlas with P. falciparum erythrocytic stages of a recently adapted isolate, and with it defined the developmental stage of over 25000 parasites from six asymptomatic children at the dry-wet season transition, and nine children with clinical malaria in the wet season. Our data confirms that older parasites circulate in the dry season, and reveals transcriptional modulation of exported proteins that affect cytoadhesion of infected erythrocytes, which we validated by immuno-fluorescence microscopy.

molecular biology↗

bioRxiv2024

Plasmodium falciparum expresses fewer var genes at lower levels during asymptomatic dry season infections than clinical malaria cases

In seasonal transmission areas, clinical malaria occurs during the wet season when mosquitoes are present, while in the dry season, malaria transmission is interrupted and clinical cases are rare. In Mali, Plasmodium falciparum can persist in low parasitaemic asymptomatic individuals through the six-month dry season and shows reduced cytoadhesion of infected erythrocytes, evidenced by the circulation of further developed parasite stages compared to clinical malaria cases. How prolonged circulation of infected erythrocytes is achieved remains unknown. Here, we explored var gene expression in subclinical infections and clinical malaria cases of Malian children, collected during the dry and wet seasons. We sequenced expressed var DBL-tags, used bioinformatic tools to predict their domain composition, binding phenotype and upstream sequence type; and determined their relationship to seasonality and clinical presentation. We found that parasites of asymptomatic infections expressed fewer var genes, with a larger proportion of var transcripts attributed to one or a few vars. In contrast, clinical cases exhibited expression of many var genes at lower proportions. We found that parasites of asymptomatic carriers expressed a mixture of CD36- and EPCR-binding PfEMP1, which changed over time. We confirmed that vars encoding CD36-binding PfEMP1 dominated in non-severe malaria cases, and found no significant difference in expressed var types between dry and wet seasons. Asymptomatic carriers were older, had higher titers of anti-P. falciparum antibodies, and broader reactivity to PfEMP1, suggesting that host immunity was the main determinant limiting var transcript variation in asymptomatic carriers. However, by RNAseq and qRT-PCR we also observed significantly higher total var transcript levels in malaria cases compared to asymptomatic carriers, suggesting that in addition to the parasites switching and the hosts immune selection of expressed var genes, parasites able to sustain long-term infections may be poised for reduced PfEMP1 expression.

microbiology↗

bioRxiv2022

Barcoding genetically distinct Plasmodium falciparum strains for comparative assessment of fitness and antimalarial drug resistance

The repeated emergence of antimalarial drug resistance in Plasmodium falciparum, including to the current frontline antimalarial artemisinin, is a perennial problem for malaria control. Nextgeneration sequencing has greatly accelerated the identification of polymorphisms in resistance-associated genes, but has also highlighted the need for more sensitive and accurate laboratory tools to profile current and future antimalarials, and to quantify the impact of drug resistance acquisition on parasite fitness. The interplay of fitness and drug response is of fundamental importance in understanding why particular genetic backgrounds are better at driving the evolution of drug resistance in natural populations, but the impact of parasite fitness landscapes on the epidemiology of drug resistance has typically been laborious to accurately quantify in the lab, with assays being limited in accuracy and throughput. Here we present a scalable method to profile fitness and drug response of genetically distinct P. falciparum strains with well-described sensitivities to several antimalarials. We leverage CRISPR/Cas9 genome-editing and barcode sequencing to track unique barcodes integrated into a non-essential gene (pfrh3). We validate this approach in multiplex competitive growth assays of three strains with distinct geographical origins. Furthermore, we demonstrate that this method can be a powerful approach for tracking artemisinin response as it can identify an artemisinin resistant strain within a mix of multiple parasite lines, suggesting an approach for scaling the laborious ring-stage survival assay (RSA) across libraries of barcoded parasite lines. Overall, we present a novel high-throughput method for multiplexed competitive growth assays to evaluate parasite fitness and drug response

microbiology↗

bioRxiv2022

Resolving drug selection and migration in an inbred South American Plasmodium falciparum population with identity-by-descent analysis

The human malaria parasite Plasmodium falciparum is globally widespread, but its prevalence varies significantly between and even within countries. Most population genetic studies in P. falciparum focus on regions of high transmission where parasite populations are large and genetically diverse, such as sub-Saharan Africa. Understanding population dynamics in low transmission settings, however, is of particular importance as these are often where drug resistance first evolves. Here, we use the Pacific Coast of Colombia and Ecuador as a model for understanding the population structure and evolution of Plasmodium parasites in small populations harboring low genetic diversity. The combination of low transmission and a high proportion of monoclonal infections means there are few outcrossing events and clonal lineages persist for long periods of time. Yet despite this, the population is evolutionarily labile and has successfully adapted to multiple drug regimes. Using 166 newly sequenced whole genomes, we measure relatedness between parasites, calculated as identity by descent (IBD), and find 17 distinct but highly related clonal lineages, six of which have persisted in the region for at least a decade. This inbred population structure is captured in more detail with IBD than with other common population structure analyses like PCA, ADMIXTURE, and distance-based trees. We additionally use patterns of intra-chromosomal IBD and an analysis of haplotypic variation to explore the role of recombination in spreading drug resistance mutations throughout the region. Two genes associated with chloroquine resistance, crt and aat1, show evidence of hard selective sweeps, while selection appears soft and/or incomplete at three other key resistance loci (dhps, mdr1, and dhfr). Overall, this work highlights the strength of IBD analyses for studying parasite population structure and resistance evolution in regions of low transmission, and emphasizes that drug resistance can evolve and spread in extremely small populations, as will occur in any region nearing malaria elimination.

evolutionary biology↗

bioRxiv2019

Quantitation of vector uptake reveals non-Poissonian transfection dynamics in Plasmodium falciparum

The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets. Reverse genetics have facilitated genome-scale knockout screens in Plasmodium berghei and Toxoplasma gondii, in which pooled transfections of multiple vectors were critical to increasing scale and throughput. These approaches have not yet been implemented in P. falciparum, mainly because the extent to which pooled transfections can be performed in this species still remains unknown. Here we use next-generation sequencing to quantitate uptake of a pool of 94 barcoded vectors. The distribution of vectors in different transfections allowed us to estimate the number of barcodes and DNA molecules taken up by the parasite population. Dilution cloning showed that single transfected parasites routinely carry as many as seven episomal barcodes, revealing an intake of multiple vectors in a highly non-uniform fashion. Transfection of non-overlapping fluorescent proteins, which allowed us to follow the dynamics of the process, confirmed the tendency for parasites to take up multiple vectors from the early stages of transfection. This finding has important implications for how reverse genetics can be scaled in P. falciparum.

microbiology↗

bioRxiv2018

Red blood cell tension controls Plasmodium falciparum invasion and protects against severe malaria in the Dantu blood group

Malaria has had a major effect on the human genome, with many protective polymorphisms such as sickle cell trait having been selected to high frequencies in malaria endemic regions1, 2. Recently, we showed that a novel blood group variant, Dantu, provides 74% protection against all forms of severe malaria in homozygous individuals3-5. This is a similar degree of protection to sickle cell trait and considerably greater than the most advanced malaria vaccine, but until now the mechanism of protection has been unknown. In the current study, we demonstrate a significant impact of Dantu on the ability of Plasmodium falciparum merozoites to invade RBCs. The Dantu variant was associated with extensive changes to the RBC surface protein repertoire, but unexpectedly the malaria protective effect did not correlate with specific RBC-parasite receptor-ligand interactions. By following invasion using video microscopy, we found a strong link between RBC tension and parasite invasion and, even in non-Dantu RBCs, identified a tension threshold above which RBC invasion did not occur. Dantu RBCs had higher average tension, meaning that a higher proportion of Dantu RBCs could not be invaded. These findings not only provide an explanation for the protective effect of Dantu against severe malaria, but also provide fresh insights into the essential process of P. falciparum parasite invasion, and how invasion efficiency varies across the heterogenous populations of RBCs that are present both within and between individuals.

genetics↗

bioRxiv2017

Capturing in vivo RNA transcriptional dynamics from the malaria parasite P. falciparum

SUMMARYTo capture the transcriptional dynamics within proliferating cells, methods to differentiate nascent transcription from pre-existing mRNAs are desired. One approach is to label newly synthesized mRNA transcripts in vivo through the incorporation of modified pyrimidines. However, the human malaria parasite, Plasmodium falciparum, is incapable of pyrimidine salvage for mRNA biogenesis. To capture cellular mRNA dynamics during Plasmodium development, we have engineered parasites that can salvage pyrimidines through the expression of a single bifunctional yeast fusion gene, cytosine deaminase/uracil phosphoribosyltransferase (FCU). We show that expression of FCU allows for the direct incorporation of thiol-modified pyrimidines into nascent mRNAs. Using developmental stage-specific promoters to express FCU-GFP enables the biosynthetic capture and in-depth analysis of mRNA dynamics from subpopulations of cells undergoing differentiation. We demonstrate the utility of this method by examining the transcriptional dynamics of the sexual gametocyte stage transition, a process that is essential to malaria transmission between hosts. We find that sexual stage commitment is governed by transcriptional reprogramming and the stabilization of a subset of essential gametocyte transcripts. This new method for biosynthetic labeling of Plasmodium mRNAs is incredibly versatile and can be used to measure transcriptional dynamics at any stage of parasite development, and thiol-modified RNAs will allow for future applications to measure RNA-protein interactions in the malaria parasite.

microbiology↗