Biology subjects

Amambua-Ngwa, A.

Publications and source records attributed to Amambua-Ngwa, A..

17 recordsLinked to original sources

bioRxiv2026

Lactylated histones mark virulence gene families in the malaria parasite Plasmodium falciparum

Epigenetic pathways have many important roles controlling virulence in human malaria parasites. Histone acetylation and methylation have been closely studied in this context but the novel epigenetic mark of lactylation has not yet been examined. Here, for the first time, we profiled lactyl-histone marks across the P. falciparum genome and found them strongly enriched at virulence genes, including genes involved in cytoadhesion and other host-cell remodelling functions. Many genes were dynamically and inducibly lactylated across the cell cycle. Thus, P. falciparum could use histone lactylation to control its virulence pathways in response to the prevailing metabolic environment in its host. We extended our chromatin profiling to parasites isolated directly from human patients, showing that here too, virulence gene families were strongly lactylated. This represents the first comprehensive profiling of P. falciparum chromatin from parasites in sub-millilitre blood samples, opening up exciting new avenues to study parasite chromatin across human disease states.

microbiology↗

bioRxiv2026

Genomic signatures of selection in Anopheles funestus reveal shared and population-specific adaptive variation across African populations

Insecticide resistance in Anopheles funestus threatens malaria vector control across sub-Saharan Africa, yet genomic signatures of selection across geographically structured populations remain poorly understood. We analysed whole-genome sequence data from 635 An. funestus mosquitoes from West (Senegal, Guinea, Nigeria), Central (Cameroon), and East Africa (Kenya) to characterise population structure and identify targets of recent positive selection. Population genomic analyses revealed strong differentiation between East African and West/Central African populations, with finer-scale structuring within West Africa. Genome-wide selection scans using H12 identified sweep regions on chromosome arms 2RL and 3RL, overlapping a cytochrome P450 cluster and the gamma-aminobutyric acid (GABA) receptor locus respectively. iSAFE prioritised candidate variants within these sweeps: non-synonymous substitutions in CYP6A14 were identified at high frequencies in Guinea, Nigeria, and Cameroon, while population-restricted variants implicated octopamine receptor genes on 2RL and the GABA receptor on 3RL. Diplotype clustering and copy number variation analyses confirmed the causal role of candidate variants, embedded within extended haplotypes of reduced heterozygosity consistent with recent positive selection. These findings demonstrate that adaptive evolution in An. funestus reflects both shared and population-specific selective processes shaped by geography and ecological context. Whereas selection on detoxification pathways appears widespread, localised signals in neuromodulatory loci, including the GABA receptor and octopamine-related genes, reveal that biological systems beyond metabolic resistance contribute to mosquito adaptation. The convergence of selective signals across these gene classes highlights neuromodulatory pathways as potential complementary targets for next-generation vector control strategies.

genomics↗

bioRxiv2026

Plasmodium falciparum invasion ligand gene transcript profiles in different populations

Plasmodium falciparum merozoites invade erythrocytes using various ligand-receptor interactions. Important ligands encoded by the eba and Rh gene families have varying expression levels in different parasite isolates, affecting their vaccine candidacy. Analyses of clinical isolates from endemic areas in Africa have indicated that most variation in these expression profiles exists within each local area, and only minor differences are seen between areas, although comparisons with non-African populations have not previously been performed. To enable this, relative transcript levels of three eba genes and five Rh genes have been analysed in new population samples, Malaysian isolates sampled from Sabah State in Borneo prior to endemic malaria elimination, and Gambian isolates, cultured under the same conditions to harvest schizonts for reverse transcription quantitative PCR assays. Significant differences between these populations were seen for three of the ligand genes, levels of eba175 being higher in Malaysia, while levels of eba181 and Rh2b were lower in Malaysia. The gene transcript profiles did not differ between single genotype and or multiple-genotype isolates. The distinctness of the Malaysian population expression profile was also supported by comparing previous data on clinical isolates from Ghana. In tests for correlation with previously determined parasite multiplication rates, eba181 transcript levels correlated positively among Malaysian isolates but not among Gambian isolates. These findings suggest that expression of three P. falciparum merozoite ligands involved in invasion may be regionally differentiated, and further analysis of Asian parasite populations would be important if vaccines based on these candidates are to be considered for future use.

microbiology↗

bioRxiv2025

Geographical variation drives adaptive equilibrium of the P. falciparum sickle-associated mutations

The recent discovery of genetic mutations in Plasmodium falciparum--the most lethal malaria parasite--that enable it to overcome the protective effects of sickle cell trait, raises fundamental questions about the underlying biological and evolutionary interactions. Here we develop a geostatistical model to compare sickle haemoglobin genotype frequencies to the Plasmodium falciparum sickle-associated alleles across global populations, and find a robust association at multiple geographical scales, implying that sickle drives positive selection for these parasite mutations. A model of parasite evolution and an analysis of local haplotype patterns suggest that key features of these mutations - that they are polymorphic in all African populations and are mutually correlated despite lying in different genome regions - are caused by geographical variation in selection pressure, and that the alleles may have been maintained by balancing selection over timescales comparable to the age of the sickle mutation itself. The predicted impact of this host-parasite interaction on disease outcomes varies widely across populations, and functional data are needed to discover the biological mechanisms involved.

genetics↗

bioRxiv2025

New insights on malaria parasite adaptation yielded by samples archived for more than 50 years

Understanding evolution of human pathogens requires looking beyond effects of recent interventions or known epidemiological changes. To study malaria parasites prior to widespread drug selection, Plasmodium falciparum genome sequences were analysed from the oldest set of archived research samples yet identified, placental blood collected in the Gambia between 1966 and 1971. Genomic complexity within infections was high, infections were genetically unrelated, and no drug resistance alleles were detected. Strong signatures of positive selection are clearly seen at multiple loci throughout the genome, most of which encode surface proteins that bind erythrocytes and are targets of acquired antibody responses. Comparison of population samples obtained over a following period of almost 50 years revealed major directional allele frequency changes at several loci apart from drug resistance genes. Exceptional changes over this time are seen at gdv1 that regulates the rate of parasite sexual conversion required for transmission, and at the unlinked Pfsa1 and Pfsa3 loci previously associated with infection of individuals with sickle-cell trait. Other affected loci encode surface and transporter proteins warranting targeted functional analyses. This identification of key long-term adaptions that have not reached equilibrium is important for understanding and potentially managing future evolution of malaria parasites.

microbiology↗

bioRxiv2024

Disparate co-evolution and prevalence of sulfadoxine and pyrimethamine resistance alleles and haplotypes at dhfr and dhps genes across Africa

Sulfadoxine-pyrimethamine (SP), despite the emergence and spread of mutations in dhfr and dhps genes associated with lower treatment efficacy, is still recommended alone or in combination by the WHO for preventive treatment in pregnant women, children and infants. Therefore, it is important to understand the evolution of P. falciparum dhfr and dhps genes. Here, we used a subset of the MalariaGEN Pf7 dataset to describe haplotype frequencies across 22 African countries, including changes over time in The Gambia, Mali, Ghana and Kenya. We show that the triple mutant of dhfr, N51I/C59R/S108N, has remained the dominant haplotype across the continent with limited evidence of additional mutations. There is greater variation for dhps, with a total of 51 different haplotypes present. The dhps resistance mutation A437G has risen close to fixation across most of Africa, although at a lower frequency in the northwest malaria-endemic part of the continent (Gambia, Senegal and Mali). The A437G mutation is usually found together with K540E in East Africa, but K540E is still very rare in West Africa. Although samples from Madagascar have low genetic differentiation from samples from mainland East Africa at the whole genome level, we show that dhps K540E is highly differentiated between the two populations, being at very low frequency in Madagascar (4%). We used whole genome data to show that only 12 SNPs are more highly differentiated than K540E between Madagascar and East Africa, with aat1 and a possible novel drug resistance locus approximately 20kb 3 of mdr1 having even higher FST. We highlight the value of longitudinal sampling and whole genome sequence data for understanding the heterogeneity and ongoing changes in anti-malarial drug resistance genetic markers.

genomics↗

bioRxiv2024

Higher multiplication rates of Plasmodium falciparum in isolates from hospital cases compared with community infections

BackgroundParasite multiplication rates vary among Plasmodium falciparum isolates from patients with malaria, suggesting differences in virulence potential, although direct comparisons between hospital-based clinical cases and community infections are needed. MethodsCryopreserved blood samples from malaria cases presenting to a district hospital in The Gambia and infections detected in local communities were introduced to continuous culture under the same conditions. Thirty-four isolates (23 hospital-based and 11 community-based) were successfully established and then tested under exponential growth conditions over six days to derive estimated P. falciparum multiplication rates per cycle based on a 48-hour typical cycle length. ResultsA range of parasite multiplication rates in culture was seen across isolates, from 1.5-fold to 5.0-fold per cycle. Multiplication rates were significantly higher in the hospital-based isolates than the community-based isolates. There was a significantly positive correlation between parasitaemia in peripheral blood and multiplication rates in culture. There was no significant difference in multiplication rates between isolates with single or multiple parasite genotypes. ConclusionsThese findings are consistent with a hypothesis that intrinsic natural variation in parasite multiplication rate may affect levels of parasitaemia achieved during infection, and that this affects likelihood of hospital presentation. Results do not support a hypothesis that parasites modify their multiplication rates in response to competing parasites with different genotypes. SummaryRelevant to understanding parasite virulence, this study finds higher Plasmodium falciparum multiplication rates in cultured isolates from malaria cases presenting to hospital than in isolates from local community infections, and positive correlation with parasitaemia in peripheral blood of individuals.

microbiology↗

bioRxiv2024

Regional Plasmodium falciparum subpopulations and malaria transmission connectivity in Africa were detected with an enlarged panel of genome-wide microsatellite loci

Unravelling the genetic diversity of Plasmodium falciparum malaria parasite provides critical information on how populations are affected by interventions and the environment, especially the evolution of molecular markers associated with parasite fitness and adaptation to drugs and vaccines. This study expands previous studies based on small sets of microsatellite loci, which often showed limited substructure in African populations of P. falciparum. Combining several short tandem repeat detection algorithms, we genotyped and analysed 2329 polymorphic microsatellite loci from next-generation sequences of 992 low-complexity P. falciparum isolates from 15 sub-Saharan African countries. Based on pairwise relatedness, we identified seven subpopulations and gene flow between the Central and Eastern African populations. The most divergent subpopulation was from Ethiopia, while unexpected unique subpopulations from Gabon and Malawi were resolved. Isolates from the Democratic Republic of Congo shared ancestry with multiple regional populations, suggesting a possible founder population of P. falciparum from the Congo basin, where there was stronger geneflow eastwards to Tanzania, and Kenya. and Malawi. The most differentiated microsatellite loci were those around the P. falciparum dihydropteroate synthase (Pfdhps) gene associated with sulphadoxine resistance. Haplotypes around the Pfdhps gene separated the West, Central, and East Africa parasite populations into distinct clusters, suggesting independent local evolution of Pfdhps-associated sulphadoxine resistance alleles in each African region. Overall, this study presents genome-wide microsatellites as markers for resolving P. falciparum population diversity, structure, and evolution in populations like Africa, where there is high gene flow.

genomics↗

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↗

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

The amino acid transporter pfaat1 modulates chloroquine resistance and fitness in malaria parasites

Malaria parasites break down host hemoglobin into peptides and amino acids in the digestive vacuole for export to the parasite cytoplasm for growth: interrupting this process is central to the mode of action of several antimalarial drugs. Mutations in the chloroquine (CQ) resistance transporter, pfcrt, located in the digestive vacuole membrane, confer CQ resistance in Plasmodium falciparum, but typically affect parasite fitness. However, the role of other parasite loci in the evolution of CQ resistance is unclear. Here we use a combination of population genomics, genetic crosses and gene editing to demonstrate that a second vacuolar transporter plays a key role in both resistance and compensatory evolution. Longitudinal genomic analyses of the Gambian parasites revealed temporal signatures of selection on an amino acid transporter (pfaat1) S258L variant which increased from 0-87% in frequency between 1984 and 2014 in parallel with pfcrt1 K76T. Parasite genetic crosses then identified a chromosome 6 quantitative trait locus containing pfaat1 that is selected by CQ treatment. Gene editing demonstrated that pfaat1 S258L potentiates CQ-resistance but at a cost of reduced fitness, while pfaat1 F313S, a common Southeast Asian polymorphism, reduces CQ-resistance while restoring fitness. Our analyses reveal hidden complexity in CQ-resistance evolution, suggesting that pfaat1 may underlie regional differences in the dynamics of resistance evolution, and modulate parasite resistance or fitness by manipulating the balance between both amino acid and drug transport.

genomics↗

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↗

bioRxiv2020

Origin of imported SARS-CoV-2 strains in The Gambia identified from whole genome sequences

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a positive-sense single stranded RNA virus with high human transmissibility. This study generated Whole Genome data to determine the origin and pattern of transmission of SARS-CoV-2 from the first six cases tested in The Gambia. Total RNA from SARS-CoV-2 was extracted from inactivated nasopharyngeal-oropharyngeal swabs of six cases and converted to cDNA following the ARTIC COVID-19 sequencing protocol. Libraries were constructed with the NEBNext ultra II DNA library prep kit for Illumina and Oxford Nanopore Ligation sequencing kit and sequenced on Illumina MiSeq and Nanopore GridION, respectively. Sequencing reads were mapped to the Wuhan reference genome and compared to eleven other SARS-CoV-2 strains of Asian, European and American origins. A phylogenetic tree was constructed with the consensus genomes for local and non-African strains. Three of the Gambian strains had a European origin (UK and Spain), two strains were of Asian origin (Japan). In The Gambia, Nanopore and Illumina sequencers were successfully used to identify the sources of SARS-CoV-2 infection in COVID-19 cases.

genomics↗

bioRxiv2020

Biting and resting preferences of malaria vectors in The Gambia

BackgroundThe scale-up of indoor residual spraying and long-lasting insecticidal nets, together with other interventions have considerably reduced the malaria burden in The Gambia. This study examined the biting and resting preferences of the local insecticide-resistant vector populations few years following scale-up of anti-vector interventions. MethodIndoor and outdoor-resting Anopheles gambiae mosquitoes were collected between July and October 2019 from ten villages in five regions in The Gambia using pyrethrum spray collection (indoor) and prokopack aspirator from pit traps (outdoor). Polymerase chain reaction assays were performed to identify molecular species, insecticide resistance mutations, Plasmodium infection rate and host blood meal. ResultsA total of 844 mosquitoes were collected both indoors (421, 49.9%) and outdoors (423, 50.1%). Four main vector species were identified, including An. arabiensis (indoor: 15%, outdoor: 26%); An. coluzzii (indoor: 19%, outdoor: 6%), An. gambiae s.s. (indoor: 11%, outdoor: 16%), An. melas (indoor: 2%, outdoor: 0.1%) and hybrids of An. coluzzii-An. gambiae (indoors: 3%, outdoors: 2%). A significant preference for outdoor resting was observed in An. arabiensis (Pearson X2=22.7, df=4, P<0.001) and for indoor resting in An. coluzzii (Pearson X2=55.0, df=4, P<0.001). Prevalence of the voltage-gated sodium channel (Vgsc)-1014S was higher in the indoor-resting (allele freq. = 0.96, 95%CI: 0.78-1) than outdoor-resting (allele freq. = 0.82, 95%CI: 0.76-0.87) An. arabiensis population. For An. coluzzii, the prevalence of most mutation markers were higher in the outdoor (allele freq. = 0.92, 95%CI: 0.81-0.98) than indoor-resting (allele freq. = 0.78, 95%CI: 0.56-0.86) mosquitoes. Sporozoite positivity rate was 1.3% (95% CI: 0.5-2%). Indoor-resting An. coluzzii had mainly fed on human blood while indoor-resting An. arabiensis, animal blood. ConclusionThe indoor-resting behavior of An. arabiensis that preferred animal blood and had low sporozoite rates, may be determined by the Vgsc-1014S mutation. Control interventions may include complementary vector control approaches such as zooprophylaxis.

molecular biology↗

bioRxiv2020

Origin of imported SARS-CoV-2 strains in The Gambia identified from Whole Genome Sequences.

genomics↗

bioRxiv2019

A modified two-color flow cytometry assay to quantify in-vitro reinvasion and determine invasion phenotypes at low Plasmodium falciparum parasitemia

Two-color flow cytometry(2cFCM) is the most accessible method for phenotyping parasite invasion. However, current protocols require samples of field isolates at [~]1% parasitemia for assay set-up, which are becoming more uncommon in low transmission settings. Current protocols, therefore, have to be adapted for low parasitemia if the method must have continued applicability in this era of elimination. Optimizing the protocol requires addressing; interference from young uninfected RBCs background fluorescence and biased phenotypes due to limited labeled RBCs availability and/or parasite density per assay. Here, we used SYBR Green I and CTFR Proliferation fluorescent dyes to set-up invasion assays with Plasmodium falciparum 3D7, Dd2 and field isolates cultures (diluted at 0.05% to 2.0% parasitemia) against varying unlabeled to labeled RBC ratios (1:1 to 1:4). We showed that a shorter SYBR Green I staining time of 20 minutes, down from 1hour, minimized background fluorescence from uninfected RBCs (mean= 0.03% events) and allowed 2cFCM to accurately quantify reinvasion for an assay at 0.02% parasitemia. An increase in labeled target RBCs to 1:3 per assays significantly increased heterologous reinvasion (p<0.001). This resulted in a 10% greater invasion inhibition by enzyme treatments (p<0.05). Strain-specific invasion phenotype could be accurately determined for samples with as low as 0.3% parasitemia. Samples above 0.8% parasitemia were less accurate. These findings show that invasion pathway phenotypes can be obtained for field samples with low parasitemia at greater sensitivity and reproducibility by increasing the proportion of labeled RBCs per assay by at least 2-fold what is in current methods.

cell biology↗

bioRxiv2019

High prevalence of Plasmodium malariae from hospital and communities in a high malaria transmission setting of Southern Nigeria

While Plasmodium falciparum continues to be the main target for malaria elimination, other Plasmodium species persist in Africa. Their clinical diagnosis is uncommon while rapid diagnostic tests (RDTs), the most widely used malaria diagnostic tool, are only able to distinguish between P. falciparum and non-falciparum species, the latter as pan-species. Blood samples, both from clinical cases and communites, were collected in southern Nigeria (Lagos and Calabar) in 2017 (October-December) and 2018 (October-November), and analysed by several methods, namely microscopy, quantitative real-time PCR (qPCR), and peptide serology targeting candidate antigens (PmAMA1, PmLDH and PmCSP). The sensitivity of the diagnostic approaches for the dominant P. falciparum were comparable, detecting approx. 80% infection, but not so for non-falciparum species - 3% and 10% infection for P. malariae; 0% and 3% infection for P. ovale by microscopy and qPCR respectively, across communities. P. ovale prevalence was less than 5%. Infection rates for P. malariae varied between age groups, with the highest rates in individuals > 5 years. P. malariae specific seroprevalence rates fluctuated in those < 10 years but generally reached the peak around 20 years of age for all peptides. The heterogeneity and rates of these non-falciparum species calls for increased specific diagnosis and targeting by elimination strategies.

microbiology↗