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Lawniczak, M.

Publications and source records attributed to Lawniczak, M..

At least 19 recordsLinked to original sources

Twenty-five new microsporidian genomes: Insights into ploidy, reproduction, and the group's pangenome

Microsporidia are single-celled, obligately intracellular eukaryotic parasites of growing importance. Many aspects of their biology remain enigmatic, partly because of difficulties in sampling, culturing, and experimentally manipulating them. Here, we continue efforts to generate microsporidian genomes from serendipitously infected organisms sequenced for biodiversity genomics initiatives, such as the Darwin Tree of Life Project. We present 25 new microsporidian genomes: 14 biologically complete, including several at (near-)chromosome level, and 11 partial. Using RNA-seq data for three published genomes we identify Prokka as a suitable ab initio annotation approach for Microsporidia. Consistently annotating all available genomes, we investigate gene-content diversity across microsporidian clades. We find that the microsporidian pangenome exhibits extreme diversity, and that current genomic data substantially undersample the group's proteome space. Neither GC content nor genome span consistently explains orthogroup number across lineages, suggesting different evolutionary pressures. We find few one-to-one orthogroups in Microsporidia, as well as few orthogroups that are both lineage-specific and conserved across most species within a given lineage. Finally, Hi-C data confirm that tetraploid Amblyosporida microsporidia occur as two equivalent diploid units. In contrast, one Enterocytozoonida tetraploid genome suggests that all four genome copies occur within a single nucleus (a 'monokaryon'), confirming a previous proposal.

genomics↗

Efficient and precise discovery of LTR-retrotransposon families using HiRepEat

Long Terminal Repeat Retrotransposons (LTR-RTs) are mobile genetic elements that replicate within host genomes using an RNA intermediate. Recent high quality genome assemblies provide new opportunities to study these repetitive elements across evolutionary timescales. We adapt the Myers O(Nd) wavefront alignment algorithm to produce a new fast LTR detection, clustering and phylogenetically-informed consensus approach for identifying LTR-RT families in new genome sequences. We present an iterative approach to identifying and removing repeats, initially tandem repeats, that we call TACO (TAndem COmpressor), and a corresponding coordinate mapping system we call SALSA (Sorted Array for Liftover of Sequence Addresses). Together these allow us to read and write in reduced genome space and search for LTR-RTs and tandem repeats in a series of operations. This greatly reduces confounding between different repeat types and allows us to uncover nested insertions. We show results from our clustering and consensus approach across the >500 genomes in the Vertebrate Genomes Project, with analysis completing in most cases in under 60 minutes with 15Gb memory and 16 cores. We also present cross-genome LTR-RT clustering across 30 Drosophila species, as well as across the VGP, providing fine-grained resolution on species-sharing patterns of LTR-RTs and revealing candidates for horizontal transfer.

bioinformatics↗

Evaluating the impact of a sample-matched reference genome on single-cell transcriptomic inferences in Plasmodium falciparum

BackgroundPlasmodium falciparum field isolates exhibit genomic variation, including copy number variation and sequence divergence. In contrast, the P. falciparum 3D7 reference genome (Pf3D7) was derived from a long-term laboratory-adapted strain and does not fully reflect the genomic variation among field isolates. The extent to which the reference genome influences RNA-seq mapping and expression inference in natural infections remains unclear. ResultsWe generated both a reference genome and single cell RNA sequencing (scRNAseq) data from a P. falciparum-infected carrier in Mali. This new ML52 assembly was annotated using Companion with Pf3D7 as the reference. scRNAseq reads from the natural infection isolate were aligned to both the Pf3D7 genome and the isolate-specific ML52 genome, followed by locus-level alignment inspection. For most conserved genes, expression inference was concordant for both references, while genes showing reference-genome-dependent differences were investigated further. Some discrepancies were attributed to mapping artefacts or to reads aligning to unplaced genomic contigs that reflected divergent haplotypes from the co-infecting strains in the naturally infected carrier. While most multigene family loci showed concordant gene expression across both references, var genes exhibited considerable mis-mapping against 3D7 var loci as well as 2/3 of var reads not mapping at all to 3D7. ConclusionscRNAseq expression inference in P. falciparum is robust for conserved genes and most multigene families when comparing to a matched vs unmatched reference genome, but the extremely polymorphic var genes require a matched assembly in order to evaluate expression. These findings highlight the importance of the reference genome for var gene studies and should be considered when interpreting transcriptomic analyses in other organisms possessing highly variable antigenic loci.

bioinformatics↗

340 dipteran genomes reveal the origin of Muller elements and sex chromosomes in Diptera

Diptera genome evolution is traditionally viewed through the lens of Drosophila melanogaster. To provide an unbiased picture of chromosome evolution in Diptera, we reconstructed six ancestral linkage groups (ALGs) and their rearrangements using 340 chromosome-level genome assemblies from 59 dipteran Families and two outgroups. Notably, Diptera ALGs are conserved in many Nematoceran lineages, while emergence of Brachycera coincided with four chromosomal fissions, which subsequently fused in various combinations. In Schizophora and relatives, a stable karyotype of five metacentric chromosomes and a chromosome homologous to the dot emerged that is remarkably stable with the notable exception of Drosophila, where the metacentric chromosomes became acrocentric Muller elements. Furthermore, our reconstruction reveals an ancient sex chromosome system associated with a small and gene-poor ALG that frequently fused to other chromosomes.

genomics↗

De novo assembly of complete Plasmodium falciparum isolate genomes using PacBio HiFi sequencing technology

Plasmodium falciparum possesses a highly structured genome with extensive sequence diversity concentrated in Variant Surface Antigen (VSA) families. These genes--var, rif, and stevor--play key roles in immune evasion and pathogenesis and are difficult to assemble using short-read sequencing technologies. Here, we applied PacBio HiFi long-read sequencing to generate high-quality de novo genome assemblies from 43 P. falciparum parasite cultures originating from community cases in The Gambia. Parasites were culture-adapted, cloned by limiting dilution where possible, and sequenced using high molecular weight DNA extracts. Assemblies from single-genotype lineages were constructed using hifiasm, producing complete chromosomal-length scaffolds with high base accuracy without requiring short-read polishing. We recovered full repertoires of var, rif, and stevor genes and classified them into known subgroups. Together, our results demonstrate that PacBio HiFi sequencing enables accurate assembly of complex P. falciparum genomes from natural infections. This work provides a valuable genomic resource for future studies of parasite evolution, transmission dynamics, and antigenic diversity, and suggests that VSA repertoires can serve as reliable proxies of genetic relatedness across infections.

genomics↗

Assessing the population genetic structure and demographic history of Anopheles gambiae and Anopheles arabiensis at island and mainland populations in Uganda: Implications for testing novel malaria vector control approaches.

Despite substantial investments in malaria control, the disease remains a major burden in sub-Saharan Africa, particularly Uganda. Novel tools such as gene drive systems are being developed to suppress malaria vector populations, but their deployment requires detailed knowledge of mosquito population genetics. We assessed the genetic structure, diversity, and demographic history of Anopheles gambiae and Anopheles arabiensis from six sites in Uganda: three islands in Lake Victoria and three mainland sites. A total of 2918 Anopheles gambiae and 173 Anopheles arabiensis were genotyped using targeted amplicon sequencing of 62 loci across coding and non-coding regions of the genome. Population structure analyses revealed clear separation between the two species but little differentiation within each species across sites. Pairwise FST values among An. gambiae populations were low (0.00054-0.028) but often significant, with mainland populations showing higher connectivity and island populations exhibiting greater isolation. Anopheles arabiensis mainland populations showed no statistically significant differentiation, suggesting panmixia. Principal Component Analysis and Bayesian clustering similarly distinguished species-level structure but no obvious substructure within sites. Mainland An. gambiae populations displayed higher nucleotide diversity than island populations, while An. arabiensis showed the lowest diversity overall. Tajimas D values were negative across sites, consistent with recent population expansions. Effective population size estimates indicated small populations at the islands (146 to 249) compared to large mainland populations (4,054 to 8,190). These findings demonstrate strong genetic differentiation between Anopheles gambiae and Anopheles arabiensis, and subtle but meaningful structure between island and mainland Anopheles gambiae populations. The reduced diversity and small effective population sizes at island sites suggest stronger genetic drift and limited gene flow, in contrast to the highly connected mainland populations. For malaria control, this contrast has direct implications. High connectivity among mainland populations may facilitate the spread of insecticide resistance alleles, while island populations, with their relative isolation and smaller sizes, may serve as suitable sites for contained field trials of gene drive strategies. This study highlights how geographic and ecological factors shape mosquito population structure and provides critical evidence for the design and monitoring of genetic-based vector control interventions.

evolutionary biology↗

On the path to reference genomes for all biodiversity: lessons learned and laboratory protocols created in the Sanger Tree of Life core laboratory over the first 2000 species

Since its inception in 2019, the Tree of Life programme at the Wellcome Sanger Institute has released high-quality, chromosomally-resolved reference genome assemblies for over 2000 species. Tree of Life has at its core multiple teams, each of which are responsible for key components of the genome engine. One of these teams is the Tree of Life core laboratory, which is responsible for processing tissues across a wide range of species into high quality, high molecular weight DNA and intact RNA, and preparing tissues for Hi-C. Here, we detail the different workflows we have developed to successfully process a wide variety of species, covering plants, fungi, chordates, protists, arthropods, meiofauna and other metazoa. We summarise our success rates and describe how to best apply and combine the suite of current protocols, which are all publicly available at protocols.io.

molecular biology↗

Single-cell transcriptional landscapes of Aedes aegypti midgut and fat body after a bloodmeal

Aedes aegypti mosquitoes are vectors for numerous arboviruses that have an increasingly substantial global health burden. Following a bloodmeal, mosquitoes experience significant physiological changes, primarily orchestrated by the midgut and fat body tissues. These changes begin with digestion and culminate in egg production. However, our understanding of those key processes at the cellular and molecular level remains limited. We have created a comprehensive cell atlas of the mosquito midgut and fat body by employing single-cell RNA sequencing and metabolomics techniques. This atlas unveils the dynamic cellular composition and metabolic adaptations that occur following a bloodmeal. Our analyses reveal highly diverse cell populations, specialized in digestion, metabolism, immunity, and reproduction. While the midgut primarily comprises enterocytes, enteroendocrine and intestinal stem cells, the fat body consists not only of trophocytes and oenocytes, but also harbors a substantial hemocyte population and a newly found fat body-yolk cell population. The fat body exhibits a complex cellular and metabolomic profile and exerts a central role in coordinating immune and metabolic processes. Additionally, an insect-specific virus, PCLV (Phasi Charoen-Like Virus) was detected in single cells, mainly in the midgut a week after the bloodmeal. These findings highlight the complexity of the mosquitos abdominal tissues, and pave the way towards the development of exquisitely refined vector control strategies consisting of genetically targeting specific cell populations and metabolic pathways necessary for egg development after a bloodmeal.

cell biology↗

A single-cell atlas of the Culex tarsalis midgut during West Nile virus infection

The mosquito midgut functions as a key interface between pathogen and vector. However, studies of midgut physiology and virus infection dynamics are scarce, and in Culex tarsalis - an extremely efficient vector of West Nile virus (WNV) - nonexistent. We performed single-cell RNA sequencing on Cx. tarsalis midguts, defined multiple cell types, and determined whether specific cell types are more permissive to WNV infection. We identified 20 cell states comprising 8 distinct cell types, consistent with existing descriptions of Drosophila and Aedes aegypti midgut physiology. Most midgut cell populations were permissive to WNV infection. However, there were higher levels of WNV RNA (vRNA) in enteroendocrine cells, suggesting enhanced replication in this population. In contrast, proliferating intestinal stem cells (ISC) had the lowest levels of vRNA, a finding consistent with studies suggesting ISC proliferation in the midgut is involved in infection control. ISCs were also found to have a strong transcriptional response to WNV infection; genes involved in ribosome structure and biogenesis, and translation were significantly downregulated in WNV-infected ISC populations. Notably, we did not detect significant WNV-infection induced upregulation of canonical mosquito antiviral immune genes (e.g., AGO2, R2D2, etc.) at the whole-midgut level. Rather, we observed a significant positive correlation between immune gene expression levels and vRNA load in individual cells, suggesting that within midgut cells, high levels of vRNA may trigger antiviral responses. Our findings establish a Cx. tarsalis midgut cell atlas, and provide insight into midgut infection dynamics of WNV by characterizing cell-type specific enhancement/restriction of, and immune response to, infection at the single-cell level. Author SummaryWest Nile virus is the leading cause of mosquito-borne disease in N. America. Cx. tarsalis is a highly competent vector of WNV that plays a central role in the transmission and maintenance of WNV in nature. It is hypothesized that the permissibility of mosquito midgut cells contributes to the midgut infection barrier and thus impacts the ability of pathogens to establish infection in a mosquito. Additionally, it is postulated that the midgut is the most important organ with respect to determining vector competence. The recent publication of the full Cx. tarsalis genome, in conjunction with the growing body of work demonstrating the successful application of single-cell RNA sequencing methodologies in insect models made it possible for us to examine the cellular composition of the Cx. tarsalis midgut, and WNV infection dynamics therein, at single-cell resolution. We found cell-type-specific differences in viral RNA levels suggesting variability in WNV replication efficiency in specific cell types, identified patterns of differential expression associated with WNV infection in specific cell populations, and characterized aspects of the innate immune response to WNV infection at the tissue and cellular level.

microbiology↗

A complex Plasmodium falciparum cryptotype circulating at low frequency across the African continent

The population structure of the malaria parasite Plasmodium falciparum can reveal underlying demographic and adaptive evolutionary processes. Here, we analyse population structure in 4,376 P. falciparum genomes from 21 countries across Africa. We identified a strongly differentiated cluster of parasites, comprising [~]1.2% of samples analysed, geographically distributed over 13 countries across the continent. Members of this cluster, named AF1, carry a genetic background consisting of a large number of highly differentiated variants, rarely observed outside this cluster, at a multitude of genomic loci distributed across most chromosomes. At these loci, the AF1 haplotypes appear to have common ancestry, irrespective of the sampling location; outside the shared loci, however, AF1 members are genetically similar to their sympatric parasites. AF1 parasites sharing up to 23 genomic co-inherited regions were found in all major regions of Africa, at locations over 7,000 km apart. We coined the term cryptotype to describe a complex common background which is geographically widespread, but concealed by genomic regions of local origin. Most AF1 differentiated variants are functionally related, comprising structural variations and single nucleotide polymorphisms in components of the MSP1 complex and several other genes involved in interactions with red blood cells, including invasion and erythrocyte antigen export. We propose that AF1 parasites have adapted to some as yet unidentified evolutionary niche, by acquiring a complex compendium of interacting variants that rarely circulate separately in Africa. As the cryptotype spread across the continent, it appears to have been maintained mostly intact in spite of recombination events, suggesting a selective advantage. It is possible that other cryptotypes circulate in Africa, and new analysis methods may be needed to identify them.

genomics↗

Polyploidy is widespread in Microsporidia

Microsporidia are obligate intracellular eukaryotic parasites with an extremely broad host range. They have both economic and public health importance. Ploidy in microsporidia is variable, with a few species formally identified as diploid and one as polyploid. Given the increase in the number of studies sequencing microsporidian genomes, it is now possible to assess ploidy levels across all currently-explored microsporidian diversity. We estimate ploidy for all microsporidian datasets available on the Sequence Read Archive (SRA) using k-mer-based analyses, and demonstrate that polyploidy is widespread in Microsporidia, and that ploidy change is dynamic in the group. Using genome-wide heterozygosity estimates, we also show that polyploid microsporidian genomes are relatively homozygous, and we discuss the implications of these findings on the timing of polyploidisation events and their origin. Author SummaryMicrosporidia are single-celled intracellular parasites, distantly related to fungi, that can infect a broad range of hosts, from humans all the way to protozoans. Exploiting the wealth of microsporidian genomic data available, we use k-mer based analyses to assess ploidy status across the group. Understanding a genomes ploidy is crucial in order to assemble it effectively, and may also be relevant for better understanding a parasites behaviour and life cycle. We show that tetraploidy is present in at least six species in Microsporidia, and that these polyploidisation events are likely to have occurred independently. We discuss why these findings may be paradoxical, given that Microsporidia, like other intracellular parasites, have extremely small, reduced genomes.

evolutionary biology↗

Chromosome evolution in Lepidoptera

Chromosomes are a central unit of genome organisation. One tenth of all described species on Earth are Lepidoptera, butterflies and moths, and these generally possess 31 holocentric chromosomes. However, a subset of lepidopteran species display dramatic variation in chromosome counts. By analysing 210 chromosomally-complete lepidopteran genomes, the largest analysis of eukaryotic chromosomal-level reference genomes to date, we show that the diverse karyotypes of extant species are derived from 32 ancestral linkage groups, which we term Merian elements. Merian elements have remained largely intact across 250 million years of evolution and diversification. Against this stable background, we identify eight independent lineages that have evaded constraint and undergone extensive reorganisation - either by numerous fissions or a combination of fusion and fission events. Outside these lineages, fusions are rare and fissions are rarer still. Fusions tend to involve small, repeat-rich Merian elements and/or the Z chromosome. Together, our results reveal the constraints on genome architecture in Lepidoptera and enable a deeper understanding of the importance of chromosomal rearrangements in shaping the evolution of eukaryotic genomes.

evolutionary biology↗

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↗

Systemic host inflammation induces stage-specific transcriptomic modification and slower maturation in malaria parasites

Maturation rate of malaria parasites within red blood cells (RBC) can be influenced by host nutrient status or circadian rhythm. Here, we observed in mice that systemic host inflammation, induced by lipopolysaccharide (LPS) conditioning or ongoing acute malaria infection, slowed the progression of a single cohort of parasites from one generation of RBC to the next. LPS-conditioning and acute infection both triggered substantial changes to the metabolomic composition of plasma in which parasites circulated. This altered plasma directly slowed parasite maturation in a manner that could not be rescued by supplementation, consistent with the presence of inhibitory factors. Single-cell transcriptomic assessment of mixed parasite populations, exposed to a short period of systemic host inflammation in vivo, revealed specific impairment in the transcriptional activity and translational capacity of trophozoites compared to rings or schizonts. Thus, we provide in vivo evidence of transcriptomic and phenotypic plasticity of asexual blood-stage Plasmodium parasites when exposed to systemic host inflammation.

microbiology↗

A single-cell atlas of Plasmodium falciparum transmission through the mosquito

Malaria parasites have a complex life cycle featuring diverse developmental strategies, each uniquely adapted to navigate specific host environments. Here we use single-cell transcriptomics to illuminate gene usage across the transmission cycle of the most virulent agent of human malaria - Plasmodium falciparum. We reveal developmental trajectories associated with the colonisation of the mosquito midgut and salivary glands and elucidate the transcriptional signatures of each transmissible stage. Additionally, we identify both conserved and nonconserved gene usage between human and rodent parasites, which point to both essential mechanisms in malaria transmission and species-specific adaptations potentially linked to host tropism. Together, the data presented here, which are made freely available via an interactive website, establish the most complete atlas of the P. falciparum transcriptional journey to date. One sentence summarySingle-cell transcriptomics of P. falciparum transmission stages highlights developmental trajectories and gene usage.

genomics↗

Artemisinin-resistant malaria parasites show enhanced transmission to mosquitoes under drug pressure

Resistance to artemisinin combination therapy (ACT) in the Plasmodium falciparum parasite is threatening to reverse recent gains in reducing global deaths from malaria. Whilst resistance manifests as delayed asexual parasite clearance in patients following ACT treatment, the phenotype can only spread geographically via the sexual cycle and subsequent transmission through the mosquito. Artemisinin and its derivatives (such as dihydroartemisinin, DHA) as well as killing the asexual parasite form are known to sterilize male, sexual-stage gametes from activation. Whether resistant parasites overcome this artemisinin-dependent sterilizing effect has not, however, been fully tested. Here, we analysed five P. falciparum clinical isolates from the Greater Mekong Subregion, each of which demonstrated delayed clinical clearance and carried known resistance-associated polymorphisms in the Kelch13 gene (PfK13var). As well as demonstrating reduced sensitivity to artemisinin-derivates in in vitro asexual growth assays, certain PfK13var isolates also demonstrated a marked reduction in sensitivity to these drugs in an in vitro male gamete activation assay compared to a sensitive control. Importantly, the same reduction in sensitivity to DHA was observed when the most resistant isolate was assayed by standard membrane feeding assays using Anopheles stephensi mosquitoes. These results indicate that ACT use can favour resistant over sensitive parasite transmission. A selective advantage for resistant parasite transmission could also favour acquisition of further polymorphisms, such as mosquito host-specificity or antimalarial partner-drug resistance in mixed infections. Favoured transmission of resistance under ACT coverage could have profound implications for the spread of multidrug resistant malaria beyond Southeast Asia. ONE SENTENCE SUMMARYArtemisinin-resistant clinical isolates can also demonstrate resistance to the transmission-blocking effects of artemisinin-based drugs, favouring resistance transmission to the mosquito.

microbiology↗

souporcell: Robust clustering of single cell RNAseq by genotype and ambient RNA inference without reference genotypes

Methods to deconvolve single-cell RNA sequencing (scRNAseq) data are necessary for samples containing a natural mixture of genotypes and for scRNAseq experiments that multiplex cells from different donors1. Multiplexing across donors is a popular experimental design with many benefits including avoiding batch effects2, reducing costs, and improving doublet detection. Using variants detected in the RNAseq reads, it is possible to assign cells to the individuals from which they arose. These variants can also be used to identify and remove cross-genotype doublet cells that may have highly similar transcriptional profiles precluding detection by transcriptional profile. More subtle cross-genotype variant contamination can be used to estimate the amount of ambient RNA in the system. Ambient RNA is caused by cell lysis prior to droplet partitioning and is an important confounder of scRNAseq analysis3. Souporcell is a novel method to cluster cells using only the genetic variants detected within the scRNAseq reads. We show that it achieves high accuracy on genotype clustering, doublet detection, and ambient RNA estimation as demonstrated across a wide range of challenging scenarios.

bioinformatics↗

The Malaria Cell Atlas: a comprehensive reference of single parasite transcriptomes across the complete Plasmodium life cycle

Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. Here we profile the single-cell transcriptomes of thousands of individual parasites, deriving the first high-resolution transcriptional atlas of the entire Plasmodium berghei life cycle. We then use our atlas to precisely define developmental stages of single cells from three different human malaria parasite species, including parasites isolated directly from infected individuals. The Malaria Cell Atlas provides both a comprehensive view of gene usage in a complex eukaryotic parasite and an open access reference data set for the study of malaria parasites.

genomics↗