Biology subjects

Awandare, G. A.

Publications and source records attributed to Awandare, G. A..

12 recordsLinked to original sources

bioRxiv2026

Competitive multiplication rate variation of Plasmodium falciparum isolates is determined by differences in intrinsic potential

Malaria parasite virulence will be impacted by naturally occurring variation in multiplication rates. Clinical isolates of Plasmodium falciparum exhibit a range of multiplication rates under exponential growth conditions in culture, but it needs to be discovered if relative multiplication rates under competitive conditions are principally defined by this underlying multiplication rate potential. Relative multiplication rates of P. falciparum lines were investigated in 14-day competition assays against a standard competitor clone HB3, with other long-term laboratory-adapted clones showing similar or higher mean per-48-hour rates ranging from 0.98 to 1.51 relative to HB3. In contrast, thirteen Ghanaian clinical isolates cultured for close to two months prior to assay had lower rates, ranging from 0.46 to 0.76 relative to HB3. Analysing the single-genotype clinical isolates and laboratory-adapted clones, there was a highly significant correlation between the competitive rates and previously determined exponential rates (Spearmans rho = 0.95, P = 0.0003), which accounted for most of the observed variance (Pearsons r2 = 0.77, P = 0.004). Testing the effect of alternative nutritional supplementation with human serum or Albumax showed a majority of parasites had consistent competitive rates under either condition, indicating that intrinsic differences determine most variation among parasites.

microbiology↗

bioRxiv2024

Addressing pandemic-wide systematic errors in the SARS-CoV-2 phylogeny

The SARS-CoV-2 genome occupies a unique place in infection biology - it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in "eyeballing" trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 4,471,579 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of June 2024, viewable at https://viridian.taxonium.org. Each genome was constructed using a novel assembly tool called Viridian (https://github.com/iqbal-lab-org/viridian), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.

bioinformatics↗

bioRxiv2024

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↗

bioRxiv2024

Detection of naturally acquired, strain-transcending antibodies against rosetting Plasmodium falciparum strains in humans

Strain-transcending antibodies against subsets of P. falciparum blood stage surface antigens could protect children from severe malaria. However, the evidence supporting the existence of such antibodies is incomplete and inconsistent. One subset of surface antigens associated with severe malaria, rosette-mediating Plasmodium falciparum Erythrocyte Membrane Protein one (PfEMP1) variants, cause infected erythrocytes to bind to uninfected erythrocytes to form clusters of cells (rosettes) that contribute to microvascular obstruction and pathology. Here, using flow cytometry of live infected erythrocytes, we tested plasma from 80 individuals living in malaria-endemic regions for IgG recognition of the surface of four P. falciparum rosetting strains. Broadly-reactive plasma samples were then used in antibody elution experiments in which intact IgG was eluted from the surface of infected erythrocytes and transferred to heterologous rosetting strains to look for strain-transcending antibodies. We found that seroprevalence (percentage of samples that recognised each strain) against allopatric rosetting strains was high in adults (58% - 93%), but lower in children (5%-30%). Strain-transcending antibodies were present in nine out of eleven eluted antibody experiments, with six of these recognising multiple heterologous rosetting parasite strains. One eluate had rosette disrupting activity against heterologous strains, suggesting PfEMP1 as the likely target of the strain-transcending antibodies. Naturally acquired strain-transcending antibodies to rosetting P. falciparum strains in humans have not been directly demonstrated previously. Their existence suggests that such antibodies could play a role in clinical protection and raises the possibility that conserved epitopes recognised by strain-transcending antibodies could be targeted therapeutically.

microbiology↗

bioRxiv2023

A fourth locus in the Plasmodium falciparum genome associated with sickle haemoglobin

BackgroundHeterozygosity for sickle haemoglobin (HbS) confers protection against severe malaria caused by the parasite Plasmodium falciparum. Recent work has suggested that this protective effect can depend on the parasite genotype: P. falciparum sickle-associated (Pfsa) variants were found disproportionately in individuals with severe malaria carrying HbS alleles in The Gambia and Kenya. Interactions between the P. falciparum genome and HbS have not previously been investigated in mild malaria cases or in Ghana. MethodsWe performed a genome-wide association analysis of P. falciparum against human {beta}-globin genotypes in a sample of 1,368 people with mild malaria in northern Ghana. ResultsWe replicated the previously identified associations with HbS at two parasite loci (Pfsa1 and Pfsa3). Pfsa2 was absent from this population. A candidate newly identified locus within the serine/ threonine kinase FIKK4.2, which we putatively term Pfsa4, was also associated with HbS; this finding replicated in a published sample from Mali. The Pfsa1-4 mutations vary widely in frequencies across Africa, are absent or very low frequency in Asia, and are highly correlated with each-other across multiple populations. We found no strong associations with haemoglobin C. ConclusionsThis study replicates previously reported sickle-associated loci in the P. falciparum genome and has produced new evidence of a potential association with sickle haemoglobin at a fourth parasite locus. Further research is needed to validate the tentative fourth locus. These findings add new complexity to the emerging picture of association between human and co-evolving malaria parasite genomes, suggesting new avenues for functional exploration.

microbiology↗

bioRxiv2022

Nanopore sequencing for real-time genomic surveillance of Plasmodium falciparum

Malaria is a global public health priority causing over 600,000 deaths annually, mostly young children living in Sub-Saharan Africa. Molecular surveillance can provide key information for malaria control, such as the prevalence and distribution of antimalarial drug resistance. However, genome sequencing capacity in endemic countries can be limited. Here, we have implemented an end-to-end workflow for P. falciparum genomic surveillance in Ghana using Oxford Nanopore Technologies, targeting antimalarial resistance markers and the leading vaccine antigen circumsporozoite protein (csp). The workflow was rapid, robust, accurate, affordable and straightforward to implement, and could be deployed using readily collected dried blood spot samples. We found that P. falciparum parasites in Ghana had become largely susceptible to chloroquine, with persistent sulfadoxine-pyrimethamine (SP) resistance, and no evidence of artemisinin resistance. Multiple Single Nucleotide Polymorphism (SNP) differences from the vaccine csp sequence were identified, though their significance is uncertain. This study demonstrates the potential utility and feasibility of malaria genomic surveillance in endemic settings using Nanopore sequencing.

genomics↗

bioRxiv2022

Genomic variation during culture-adaptation of genetically complex Plasmodium falciparum clinical isolates

Experimental studies on the biology of malaria parasites have been mostly based on laboratory-adapted lines, but there is limited understanding of how these may differ from parasites in natural infections. Loss-of-function mutants have previously been shown to emerge during culture of some Plasmodium falciparum clinical isolates, in analyses that focused on single-genotype infections. The present study included a broader array of isolates, mostly representing multiple-genotype infections which are more typical in areas where malaria is highly endemic. Genome sequence data from multiple time points during several months of culture adaptation of 28 West African isolates were analysed, including previously available sequences along with new genome sequences from additional isolates and timepoints. Some genetically complex isolates eventually became fixed over time to single surviving genotypes in culture, whereas others retained diversity although proportions of genotypes varied over time. Drug-resistance allele frequencies did not show overall directional changes, suggesting that resistance-associated costs are not the main causes of fitness differences among parasites in culture. Loss-of-function mutants emerged during culture in several of the multiple-genotype isolates, affecting genes (including AP2-HS, EPAC and SRPK1) for which loss-of-function mutants were previously seen to emerge in single-genotype isolates. Parasite clones were derived by limiting dilution from six of the isolates, and sequencing identified de novo variants not detected in the bulk isolate sequences. Interestingly, most of these were nonsense mutants and frameshifts disrupting the coding sequence of EPAC, the gene with the largest number of independent nonsense mutants previously identified in laboratory-adapted lines. Analysis of Identity-By-Descent to explore relatedness among clones revealed co-occurring non-identical sibling parasites, illustrative of the natural genetic structure within parasite populations.

microbiology↗

bioRxiv2022

Transcriptome analysis of diverse Plasmodium falciparum clinical isolates identifies genes correlating with highly variable expression of merozoite surface protein MSPDBL2

The merozoite surface protein MSPDBL2 of Plasmodium falciparum is under strong balancing selection and is a target of naturally acquired antibodies. Remarkably, MSPDBL2 is expressed in only a minority of mature schizonts of any cultured parasite line, and mspdbl2 gene transcription increases in response to overexpression of the gametocyte development inducer GDV1, so it is important to understand its natural expression. Here, MSPDBL2 in mature schizonts was analysed in the first ex vivo culture cycle of 96 clinical isolates from four populations with varying levels of infection endemicity in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2 (median of 0.6% overall), but the frequency distribution was highly skewed as nine isolates had more than 3% schizonts positive and one had 73% positive. To investigate whether expression of other gene loci correlated with MSPDBL2 expression, whole transcriptome sequencing was performed on schizont-enriched material from 17 of the clinical isolates with a wide range of proportions of schizonts positive. Transcripts of particular parasite genes were highly significantly positively correlated with MSPDBL2 positivity in schizonts as well as with mspdbl2 gene transcript levels, with overrepresentation of genes previously implicated as likely to be involved in gametocytogenesis, but not including the gametocytogenesis master regulator ap2g. Although MSPDBL2 is apparently not directly involved in sexual commitment, it marks a co-occurring developmental subpopulation that may be functionally distinct within blood stage infections.

microbiology↗

bioRxiv2022

Variation in Plasmodium falciparum sexual commitment rates and responses to environmental modification

Asexual blood stage malaria parasites must produce sexual progeny in order to infect mosquitoes. It is therefore important to understand the scope of variation in sexual commitment rates of the most important human parasite Plasmodium falciparum, and to assess responses to conditions that may affect this commitment. First, using a genetically modified line with inducible elevated sexual commitment we compared two different methods of measuring the rates, either staining the Pfs16 marker in very early gametocytes or counting gametocytes after further development. The methods showed correlated estimates in evaluation across multiple independent replicate assays, with higher sensitivity and precision being achieved by Pfs16 marker detection in early gametocytes, so this method was used to survey a wide range of P. falciparum lines. A panel of six recently culture-established clinical isolates from Ghana showed mean sexual commitment rates per cycle ranging from 3.3% to 12.2%, with some significant differences among isolates as well as variation among biological replicates of each. Comparing 13 long-term laboratory-adapted lines of diverse origins, eight had sexual commitment rates similar to those of the recent clinical isolates, with means across multiple biological replicates for each line ranging from 4.7% to 14.5%, while four lines had significantly lower rates with means ranging from 0.3 to 1.6%, and one line with a non-functional ap2-g gene always showed zero sexual commitment. Among a subset of parasite lines, adding choline to suppress commitment had quantitatively variable effects, although these were significant in most assays of lines that had relatively high rates. This study indicates the importance of multiple assay replicates and comparisons of diverse isolates to understand natural and culture-induced variation in this key reproductive trait, relevant to investigating potential effects of parasite adaptation on transmission. Author SummaryOnly sexual malaria parasites are transmitted from humans to mosquitoes, so it is vital to understand variation in sexual commitment rates of blood stage malaria parasites, and responses to conditions that affect this. We compared two different methods of measuring the rates in a Plasmodium falciparum line with engineered sexual commitment variation, demonstrating higher sensitivity and precision by detection of an early differentiation marker, and this method was then used to survey diverse P. falciparum lines in culture with extensive biological replicate measurements. Recent clinical isolates from Ghana showed mean sexual commitment rates per cycle ranging from 3% to 12%, with significant differences among isolates as well as variation among biological replicates. Long-term laboratory-adapted lines of diverse origins had a wide range, most having rates similar to those of the clinical isolates, while a minority consistently had much lower or zero rates. There was quantitative variation among lines in the effects of adding choline to suppress commitment, although most assays of lines that had relatively high rates showed significant effects. Performing multiple biological replicates and comparisons of recent parasite isolates is vital to understand intra-specific variation in this important reproductive trait, and move towards investigating direct effects on disease transmission.

microbiology↗

bioRxiv2020

Intrinsic multiplication rate variation and plasticity of human blood stage malaria parasites

Pathogen multiplication rate is theoretically an important determinant of virulence, although often poorly understood. We show intrinsic multiplication rate variation of the major human malaria parasite Plasmodium falciparum to be associated with blood-stage infection intensity. A panel of clinical isolates from a highly endemic West African population was analysed repeatedly during five months of continuous culture, showing a range of exponential multiplication rates at all timepoints tested, mean rates increasing over time. All isolates had different genome sequences, many containing within-isolate diversity that decreased over time, but increases in multiplication rates were not primarily attributable to genomic selection. New mutants, including premature stop codons emerging in a few isolates, did not attain sufficiently high frequencies to substantially affect overall multiplication rates. Significantly, multiplication rate variation at each of the cultured timepoints robustly correlated with parasite levels in patients at clinical presentation, indicating parasite control of multiplication that contributes to virulence.

microbiology↗

bioRxiv2020

Blood donor variability is a modulatory factor for P. falciparum invasion phenotyping assays

Human erythrocytes are indispensable for Plasmodium falciparum development. Unlike other eukaryotic cells, there is no existing erythroid cell line capable of supporting long-term P. falciparum in vitro experiments. Consequently, invasion phenotyping experiments rely on erythrocytes of different backgrounds. However, the contribution of the erythrocytes variation in influencing invasion rates remains unknown, which presents a challenge for conducting large-scale comparative studies. Here, we used erythrocytes of different blood groups harboring different hemoglobin genotypes to assess the relative contribution of blood donor variability in P. falciparum invasion phenotyping assays. For each donor, we investigated the relationship between parasite invasion phenotypes and erythrocyte phenotypic characteristics, including; the expression levels of surface receptors (e.g. the human glycophorins A and C, the complement receptor 1 and decay accelerating factor), blood groups (e.g. ABO/Rh system), and hemoglobin genotypes (e.g. AA, AS and AC). Across all donors, there were significant differences in invasion efficiency following treatment with either neuraminidase, trypsin or chymotrypsin relative to the control erythrocytes. Primarily, we showed that the levels of key erythrocyte surface receptors and their sensitivity to enzyme treatment, significantly differed across donors. However, invasion efficiency correlated neither with susceptibility to enzyme treatment nor with the levels of the selected erythrocyte surface receptors. Upon further analysis, we found no relationship between P. falciparum invasion phenotype and blood group or hemoglobin genotype. ImportanceAssays to decipher P. falciparum invasion phenotypes are of great importance in the quest for an efficient malaria vaccine. Malaria associated mortality is mainly attributed to the blood stage of the parasites life cycle, a major focus of vaccine development strategies. Further, testing and validating blood stage vaccines necessitates conducting large-scale studies in endemic countries. However, comparing results from such studies is challenged by the lack of standard assays. As human erythrocytes play a pivotal role in P. falciparum invasion assays, the need to investigate the effect of blood donor variability in the outcome of such assays is apparent. The significance of our study is in reporting the absence of relationship between P. falciparum invasion efficiency and commonly shared erythrocyte features across different erythrocyte donors, therefore emphasizing the need to consider erythrocyte donor uniformity and to anticipate challenges associated to blood donor variability in early stages of large-scale study design.

microbiology↗

bioRxiv2018

Plasmodium falciparum mature schizont transcriptome variation among clinical isolates and laboratory-adapted clones

Malaria parasite genes exhibit variation in both sequence and expression level. There is much information on sequence polymorphism, but less resolution on natural variation in transcriptomes of parasites at specific developmental stages. This is largely because it is challenging to obtain highly replicated sampling of transcriptomes to overcome potentially confounding technical and biological variation. We address the issue in the major human parasite Plasmodium falciparum by obtaining RNA-seq profiles of multiple independent replicate preparations of mature schizont-stage parasites from a panel of clinical isolates recently established in culture and from long-term laboratory-adapted clones. With a goal of robustly identifying variably expressed genes, we show that increasing the numbers of biological sample replicates greatly improves the discovery rate. Generally, six independent replicates of each parasite culture is recommendable as being significantly to lower numbers, although for highly expressed genes variable expression can be detected when fewer replicates are available. A broad comparison identifies genes differing in relative expression between cultured clinical isolates and laboratory-adapted clones. Genes more highly expressed in the laboratory-adapted clones include an AP2 transcription factor gene Pf3D7_0420300 and putative methyl transferase genes. The variable expression of several known merozoite invasion ligands is confirmed, and previously uncharacterised genes are shown to be differentially expressed among clinical isolates. New RT-qPCR assays validate the variation in transcript levels of these genes, and allow quantitation of expression to be extended to a wider panel of clinical isolate samples. These variably expressed genes are new candidates for investigation as potential determinants of alternative parasite developmental pathways or targets of immunity.\n\nAuthor summaryUnderstanding parasite diversity and adaptation may require characterisation of gene expression variation, and is vital if chemotherapeutic or vaccine development is to consider new candidate targets, but it is technically challenging to generate precise data on clinical isolates. Here, we analyse the transcriptomes of mature Plasmodium falciparum schizonts using RNA-sequencing, using large numbers of biological replicate samples to minimise the impact of inter-replicate variation on observed patterns of differential expression. This identifies genes that are differentially expressed in long term laboratory-adapted parasites and recently cultured clinical isolates, as well as among different clinical isolates. In additional samples of schizonts grown in the first cycle ex vivo prior to any erythrocyte invasion, expression levels of a selected panel of these genes vary among isolates, but mean levels are similar to those in the continuously cultured clinical isolates, indicating that the latter are useful for experimental studies requiring biological replication.

cell biology↗