Biology subjects

Guler, J. L.

Publications and source records attributed to Guler, J. L..

11 recordsLinked to original sources

bioRxiv2026

Environmental stress promotes entry into a pre-existing latent state in Plasmodium falciparum

Artemisinin treatment eliminates most Plasmodium falciparum malaria parasites but leaves behind a small population capable of long-term survival and eventual recrudescence. Although these survivors have been associated with latency, it remains unclear whether drug treatment induces this state or selectively enriches parasites already predisposed for persistence. Environmental stress also enhances parasite survival following artemisinin exposure, suggesting that stress may promote persistence by increasing entry into latency. Here, we show that environmental stress promotes entry into latency and define the molecular features of this state using single-cell transcriptomics. Mild nutrient deprivation increased latency formation 2- to 3-fold following dihydroartemisinin (DHA) exposure, and latency frequency positively correlated with long-term parasite recovery. Single-cell RNA sequencing revealed a transcriptionally distinct early trophozoite population that emerged after DHA exposure and diverged from the normal developmental trajectory. A 200-gene classifier confirmed strong enrichment of this signature following artemisinin exposure across independent datasets. The classifier also detected rare latent-like parasites in untreated populations, suggesting the presence of a rare parasite subpopulation already predisposed for persistence. Consistent with previous studies, latent parasites exhibited reduced investment in growth-associated functions; however, our analyses revealed that latency is accompanied by extensive and coordinated transcriptional remodeling; they selectively maintained mitochondrial functions, redox homeostasis, nutrient acquisition pathways, lipid metabolism, and stress-responsive signaling. Latent parasites also preserved invasion functions and host-cell interactions, with many of the strongest latency markers encoding exported proteins associated with the erythrocyte and Maurer's clefts. Environmental stress increased entry into this state without substantially altering its core transcriptional architecture, suggesting that stress influences the decision to enter latency more than the biology of latency itself. These findings support a model in which artemisinin survival arises, in part, through enrichment of a pre-existing latent subpopulation and identify promising candidates to target latency for future antimalarial interventions.

microbiology↗

bioRxiv2025

SVCROWS: A User-Defined Tool for Delineating Biologically Significant Structural Variants in Heterogeneous Datasets

Genomic structural variants (SVs) are pervasive and can impose major phenotypic impacts. However, it is difficult to appreciate the individual significance of SVs when they are heterogeneously positioned across a genomic neighborhood. Further, ubiquitous variance in SV calling accuracy complicates SV counting and downstream analysis. Tools exist to simplify SV datasets but they are not suited for all applications. Here, we present a new SV merger, SVCROWS: Structural Variation Consensus with Reciprocal Overlap and Weighted Sizes. This option-rich merger summarizes SV regions using a size-weighted reciprocal overlap framework, accounting for skewed impacts of variable-length SVs. User input directs stringency, enabling various levels of resolution in complex genome regions that harbor a spectrum of SV sizes. Further, by optimizing SVCROWS parameters, the user can tailor results to their study system. When compared to other SV merging programs, SVCROWS maintained accuracy and conserved rare genotypes from both simulated and real-world datasets. Visualization of merger output was critical for identifying how some algorithms derived erroneous conclusions while SVCROWS remained reliable, especially in complex regions. Overall, the novel SVCROWS algorithm presents an improved framework for SV interpretation; its intuitive nature and generalizability facilitates its application to virtually any workflow. Graphical AbstractSVCROWS (Structural Variation Consensus with Reciprocal Overlap and Weighted Sizes) is a structural variant merger that leverages option-rich, size-weighted comparisons to better resolve complex inputs and separate out regions of meaningful biological differences. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/634734v3_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1658d18org.highwire.dtl.DTLVardef@1239f0org.highwire.dtl.DTLVardef@99efeaorg.highwire.dtl.DTLVardef@5607a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

HapCNV: A Comprehensive Framework for CNV Detection in Low-input DNA Sequencing Data

Copy number variants (CNVs) are prevalent in both diploid and haploid genomes, with the latter containing a single copy of each gene. Studying CNVs in genomes from single or few cells is significantly advancing our knowledge in human disorders and disease susceptibility. Low-input including low-cell and single-cell sequencing data for haploid and diploid organisms generally displays shallow and highly non-uniform read counts resulting from the whole genome amplification steps that introduce amplification biases. In addition, haploid organisms typically possess relatively short genomes and require a higher degree of DNA amplification compared to diploid organisms. However, most CNV detection methods are specifically developed for diploid genomes without specific consideration of effects on haploid genomes. Challenges also reside in reference samples or normal controls which are used to provide baseline signals for defining copy number losses or gains. In traditional methods, references are usually pre-specified from cells that are assumed to be normal or disease-free. However, the use of pre-defined reference cells can bias results if common CNVs are present. Here, we present the development of a comprehensive statistical framework for data normalization and CNV detection in haploid single- or low-cell DNA sequencing data called HapCNV. The prominent advancement is the construction of a novel genomic location specific pseudo-reference that selects unbiased references using a preliminary cell clustering method. This approach effectively preserves common CNVs. Using simulations, we demonstrated that HapCNV outperformed existing methods by generating more accurate CNV detection, especially for short CNVs. Superior performance of HapCNV was also validated in detecting known CNVs in a real P. falciparum parasite dataset. In conclusion, HapCNV provides a novel and useful approach for CNV detection in haploid low-input sequencing datasets, with easy applicability to diploids.

bioinformatics↗

bioRxiv2024

Replication stress increases de novo CNVs across the malaria parasite genome

Changes in the copy number of large genomic regions, termed copy number variations (CNVs), contribute to important phenotypes. CNVs are readily identified using conventional approaches when present in a large fraction of the cell population. However, CNVs in only a few genomes are often overlooked but important; if beneficial, a de novo CNV that arises in a single genome can expand during selection to create a population of cells with novel characteristics. While single cell methods for studying de novo CNVs are increasing, we continue to lack information about CNV dynamics in rapidly evolving microbial populations. Here, we investigated de novo CNVs in the genome of the Plasmodium parasite that causes human malaria. The highly AT-rich P. falciparum genome readily accumulates CNVs that facilitate rapid adaptation. We employed low-input genomics and specialized computational tools to evaluate the impact of sub-lethal stress on the de novo CNV rate. We observed a significant increase in genome-wide de novo CNVs following treatment with an antimalarial compound that inhibits replication. De novo CNVs encompassed genes from various cellular pathways participating in human infection. This snapshot of CNV dynamics emphasizes the connection between replication stress, DNA repair, and CNV generation in this important microbial pathogen.

genomics↗

bioRxiv2023

Expansion of GTP cyclohydrolase I copy number in malaria parasites resistant to a pyrimidine biosynthesis inhibitor

Increases in the copy number of large genomic regions, termed genome amplification, are an important adaptive strategy for malaria parasites. Numerous amplifications across the Plasmodium falciparum genome contribute directly to drug resistance or impact the fitness of this protozoan parasite. During the characterization of parasite lines with amplifications of the dihydroorotate dehydrogenase (DHODH) gene, we detected increased copies of an additional genomic region that encompassed 3 genes (~5 kb) including GTP cyclohydrolase I (GCH1 amplicon). While this gene is reported to increase the fitness of antifolate resistant parasites, GCH1 amplicons had not previously been implicated in any other antimalarial resistance context. Here, we further explored the association between GCH1 and DHODH copy number. Using long read sequencing and single read visualization, we directly observed a higher number of tandem GCH1 amplicons in parasites with increased DHODH copies (up to 9 amplicons) compared to parental parasites (3 amplicons). While all GCH1 amplicons shared a consistent structure, expansions arose in 2-unit steps (from 3 to 5 to 7, etc copies). Adaptive evolution of DHODH and GCH1 loci was further bolstered when we evaluated prior selection experiments; DHODH amplification was only successful in parasite lines with pre-existing GCH1 amplicons. These observations, combined with the direct connection between metabolic pathways that contain these enzymes, lead us to propose that the GCH1 locus is beneficial for the fitness of parasites exposed to DHODH inhibitors. This finding highlights the importance of studying variation within individual parasite genomes as well as biochemical connections of drug targets as novel antimalarials move towards clinical approval. Author SummaryMalaria is caused by a protozoan parasite that readily evolves resistance to drugs that are used to treat this deadly disease. Changes that arise in the parasite genome, including extra copies of important genes, directly contribute to this resistance or improve how well the resistant parasite competes. In this study, we identified that extra copies of one gene (GTP cyclohydrolase or GCH1) were more likely to be found in parasites with extra copies of another gene on a different chromosome (dihydroorotate dehydrogenase or DHODH). A method that allows us to view long pieces of DNA from individual genomes was especially important for this study; we were able to assess gene number, arrangement, and boundary sequences, which provided clues into how extra copies evolved. Additionally, by analyzing previous experiments, we identified that extra GCH1 copies improved resistance to drugs that target DHODH. The relationship between these two loci is supported by a direct connection between the folate and pyrimidine biosynthesis pathways that the parasite uses to make DNA. Since GCH1 amplicons are common in clinical parasites worldwide, this finding highlights the need to study metabolic connections to avoid resistance evolution.

genomics↗

bioRxiv2022

Nutrient limitation mimics artemisinin tolerance in malaria

Mounting evidence demonstrates that nutritional environment can alter pathogen drug sensitivity. While the nutrient-rich media used for standard in vitro culture contains supra-physiological nutrient concentrations, pathogens encounter a relatively restrictive environment in vivo. We assessed the effect of nutrient limitation on the protozoan parasite that causes malaria and demonstrated that short-term growth under physiologically-relevant mild nutrient stress (or "metabolic priming") triggers increased tolerance of the potent antimalarial drug dihydroartemisinin (DHA). We observed beneficial effects using both short-term survival assays and longer-term proliferation studies, where metabolic priming increases parasite survival to a level previously defined as DHA resistant (>1% survival). We performed these assessments by either decreasing single nutrients that have distinct roles in parasite metabolism or using a media formulation with reductions in many nutrients that simulates the human plasma environment. We determined that priming-induced DHA tolerance was restricted to parasites that had newly invaded the host red blood cell but the effect was not dependent on genetic background. The molecular mechanisms of this intrinsic effect mimic aspects of genetic artemisinin tolerance, including translational repression, autophagy, and protein export. This finding suggests regardless of the impact on survival rates, environmental stress could stimulate changes that ultimately directly contribute to drug tolerance. Because metabolic stress is likely to occur more frequently in vivo compared to the stable in vitro environment, priming-induced drug tolerance has ramifications for how in vitro results translate to in vivo studies. Improving our understanding of how pathogens adjust their metabolism to impact survival of current and future drugs is an important avenue of research to prevent and slow the spread of resistance.

microbiology↗

bioRxiv2020

Single cell sequencing of the small and AT-skewed genome of malaria parasites

Single cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. Here, we present a single cell sequencing pipeline for the intracellular parasite, Plasmodium falciparum, which harbors a relatively small genome with an extremely skewed base content. Through optimization of a quasi-linear genome amplification method, we achieve better targeting of the parasite genome over contaminants and generate coverage levels that allow detection of relatively small copy number variations on a single cell level. These improvements are important for expanding accessibility of single cell approaches to new organisms and for improving the study of adaptive mechanisms.

genomics↗

bioRxiv2019

Comparative analyses of parasites with a comprehensive database of genome-scale metabolic models

Protozoan parasites cause diverse diseases with large global impacts. Research on the pathogenesis and biology of these organisms is limited by economic and experimental constraints. Accordingly, studies of one parasite are frequently extrapolated to infer knowledge about another parasite, across and within genera. Model in vitro or in vivo systems are frequently used to enhance experimental manipulability, but these systems generally use species related to, yet distinct from, the clinically relevant causal pathogen. Characterization of functional differences among parasite species is confined to post hoc or single target studies, limiting the utility of this extrapolation approach. To address this challenge and to accelerate parasitology research broadly, we present a functional comparative analysis of 192 genomes, representing every high-quality, publicly-available protozoan parasite genome including Plasmodium, Toxoplasma, Cryptosporidium, Entamoeba, Trypanosoma, Leishmania, Giardia, and other species. We generated an automated metabolic network reconstruction pipeline optimized for eukaryotic organisms. These metabolic network reconstructions serve as biochemical knowledgebases for each parasite, enabling qualitative and quantitative comparisons of metabolic behavior across parasites. We identified putative differences in gene essentiality and pathway utilization to facilitate the comparison of experimental findings. This knowledgebase represents the largest collection of genome-scale metabolic models for both pathogens and eukaryotes; with this resource, we can predict species-specific functions, contextualize experimental results, and optimize selection of experimental systems for fastidious species.

systems biology↗

bioRxiv2018

SLOPE: A two-part method for the enrichment of ring stage Plasmodium falciparum parasites

Plasmodium protozoan parasites undergo rounds of asexual replication inside human erythrocytes, progressing from ring stage, to trophozoites and schizonts, before egress and reinvasion. Given the discovery of ring-specific artemisinin tolerance and quiescence in Plasmodium falciparum, there is great urgency to better understand ring stage biology. However, the lack of an effective enrichment method has left rings and related parasite stages understudied compared to their late stage counterparts, which can be easily isolated due to their paramagnetic properties. Here, a method for separating all Plasmodium infected erythrocytes from uninfected erythrocytes is presented. This approach takes advantage of streptolysin-O (SLO) to preferentially lyse uninfected erythrocytes as previously shown by Jackson, et al. Following lytic treatment, Percoll gradient centrifugation removes lysed cells, leaving an intact cell population enriched in infected erythrocytes. This SLO-Percoll (SLOPE) method is effective on stages from the entire erythrocytic cycle, including previously inaccessible forms such as circulating rings from malaria-infected patients and artemisinin-induced quiescent parasites. Furthermore, the utility of SLOPE is extended to multiple media formulations used for the propagation of two human Plasmodium species. The alteration of external cholesterol levels modulates SLOPE effectiveness, demonstrating the role of erythrocyte membrane cholesterol in lytic discrimination. Importantly, enrichment does not impact parasite viability, which establishes the non-toxic nature of SLOPE. Targeted metabolomics of SLOPE-enriched ring stage samples confirms the impact on treated samples; parasite-derived metabolites are increased and contaminating host material is reduced compared to non-enriched samples.\n\nImportanceMalaria is caused by infection with protozoan Plasmodium parasites and is responsible for over 400,000 deaths annually. The availability of effective antimalarial drugs is critical to the reduction of malaria-related mortality, yet widespread resistance highlights the need for the continued study of Plasmodium biology. The SLOPE method is an accessible, scalable, rapid (30-40min), and non-toxic enrichment method that is broadly effective on many erythrocytic stages. This method is ideal for use upstream of a variety of sensitive analyses, which will increase experimental quality in virtually all areas of asexual Plasmodium parasite research. Further, because the consumption of cholesterol is a common characteristic of other intracellular parasites (both bacteria and other protozoa), SLOPE holds potential for extension to other relevant pathogens.

microbiology↗

bioRxiv2018

Leveraging the effects of chloroquine on resistant malaria parasites for combination therapies

Malaria is a major global health problem, with the Plasmodium falciparum protozoan parasite causing the most severe form of the disease. Prevalence of drug-resistant P. falciparum highlights the need to understand the biology of resistance and to identify novel combination therapies that are effective against resistant parasites. Resistance has compromised the therapeutic use of many antimalarial drugs, including chloroquine, and limited our ability to treat malaria across the world. Fortunately, chloroquine resistance comes at a fitness cost to the parasite; this can be leveraged in developing combination therapies or to reinstate use of chloroquine. To understand biological changes induced by chloroquine treatment, we compared transcriptomics data from chloroquine-resistant parasites in the presence or absence of the drug. Using both linear models and a genome-scale metabolic network reconstruction of the parasite to interpret the expression data, we identified targetable pathways in resistant parasites. This study identified an increased importance of lipid synthesis, glutathione production/cycling, isoprenoids biosynthesis, and folate metabolism in response to chloroquine. We identified potential drug targets for chloroquine combination therapies. Significantly, our analysis suggests that the combination of chloroquine and sulfadoxine-pyrimethamine or fosmidomycin may be more effective against chloroquine-resistant parasites than either drug alone; further studies will explore the use of these drugs as chloroquine resistance blockers. Additional metabolic weaknesses were found in glutathione generation and lipid synthesis during chloroquine treatment. These processes could be targeted with novel inhibitors to reduce parasite growth and reduce the burden of malaria infections. Thus, we identified metabolic weaknesses of chloroquine-resistant parasites and propose targeted chloroquine combination therapies.

microbiology↗

bioRxiv2017

Methods matter: Influential purification and analysis parameters for intracellular parasite metabolomics

Metabolomics is increasingly popular for the study of many pathogens. For the malaria parasite, Plasmodium falciparum, both targeted and untargeted metabolite detection has improved our understanding of pathogenesis, host-parasite interactions, and antimalarial drug treatment and resistance. However, purification and analysis procedures for performing metabolomics on intracellular pathogens have not been explored. Here, we investigate the impact of host contamination on the metabolome when preparing samples using standard methods. We purified in vitro grown ring stage intra-erythrocytic P. falciparum parasites for untargeted metabolomics studies; the small size of this developmental stage amplifies the challenges associated with metabolomics studies as the ratio between host and parasite biomass is maximized. Following metabolite identification and data preprocessing, we investigated whether host contributions could be corrected post hoc using various normalization approaches (including double stranded DNA, total protein, or parasite number). We conclude that normalization parameters have large effects on differential abundance analysis and recommend the thoughtful selection of these parameters. However, normalization does not remove the contribution from the parasites extracellular environment (culture media and host erythrocyte). In fact, we found that extra-parasite material is as influential on the metabolome as treatment with a potent antimalarial drug with known metabolic effects (artemisinin). Because of this influence, we could not detect significant changes associated with drug treatment. Instead, we identified metabolites predictive of host and media contamination that can be used to assess sample purification. Our findings provide a basis for development of improved experimental and analytical methods for future metabolomics studies of intracellular organisms.

microbiology↗