Publications

2014

Nair, Shalini, Standwell C Nkhoma, David Serre, Peter A Zimmerman, Karla Gorena, Benjamin J Daniel, Francois Nosten, Timothy J C Anderson, and Ian H Cheeseman. (2014) 2014. “Single-Cell Genomics for Dissection of Complex Malaria Infections.”. Genome Research 24 (6): 1028-38. https://doi.org/10.1101/gr.168286.113.

Most malaria infections contain complex mixtures of distinct parasite lineages. These multiple-genotype infections (MGIs) impact virulence evolution, drug resistance, intra-host dynamics, and recombination, but are poorly understood. To address this we have developed a single-cell genomics approach to dissect MGIs. By combining cell sorting and whole-genome amplification (WGA), we are able to generate high-quality material from parasite-infected red blood cells (RBCs) for genotyping and next-generation sequencing. We optimized our approach through analysis of >260 single-cell assays. To quantify accuracy, we decomposed mixtures of known parasite genotypes and obtained highly accurate (>99%) single-cell genotypes. We applied this validated approach directly to infections of two major malaria species, Plasmodium falciparum, for which long term culture is possible, and Plasmodium vivax, for which no long-term culture is feasible. We demonstrate that our single-cell genomics approach can be used to generate parasite genome sequences directly from patient blood in order to unravel the complexity of P. vivax and P. falciparum infections. These methods open the door for large-scale analysis of within-host variation of malaria infections, and reveal information on relatedness and drug resistance haplotypes that is inaccessible through conventional sequencing of infections.

2012

Amambua-Ngwa, Alfred, Kevin K A Tetteh, Magnus Manske, Natalia Gomez-Escobar, Lindsay B Stewart, Elizabeth Deerhake, Ian H Cheeseman, et al. (2012) 2012. “Population Genomic Scan for Candidate Signatures of Balancing Selection to Guide Antigen Characterization in Malaria Parasites.”. PLoS Genetics 8 (11): e1002992. https://doi.org/10.1371/journal.pgen.1002992.

Acquired immunity in vertebrates maintains polymorphisms in endemic pathogens, leading to identifiable signatures of balancing selection. To comprehensively survey for genes under such selection in the human malaria parasite Plasmodium falciparum, we generated paired-end short-read sequences of parasites in clinical isolates from an endemic Gambian population, which were mapped to the 3D7 strain reference genome to yield high-quality genome-wide coding sequence data for 65 isolates. A minority of genes did not map reliably, including the hypervariable var, rifin, and stevor families, but 5,056 genes (90.9% of all in the genome) had >70% sequence coverage with minimum read depth of 5 for at least 50 isolates, of which 2,853 genes contained 3 or more single nucleotide polymorphisms (SNPs) for analysis of polymorphic site frequency spectra. Against an overall background of negatively skewed frequencies, as expected from historical population expansion combined with purifying selection, the outlying minority of genes with signatures indicating exceptionally intermediate frequencies were identified. Comparing genes with different stage-specificity, such signatures were most common in those with peak expression at the merozoite stage that invades erythrocytes. Members of clag, PfMC-2TM, surfin, and msp3-like gene families were highly represented, the strongest signature being in the msp3-like gene PF10_0355. Analysis of msp3-like transcripts in 45 clinical and 11 laboratory adapted isolates grown to merozoite-containing schizont stages revealed surprisingly low expression of PF10_0355. In diverse clonal parasite lines the protein product was expressed in a minority of mature schizonts (<1% in most lines and ∼10% in clone HB3), and eight sub-clones of HB3 cultured separately had an intermediate spectrum of positive frequencies (0.9 to 7.5%), indicating phase variable expression of this polymorphic antigen. This and other identified targets of balancing selection are now prioritized for functional study.

Nkhoma, Standwell C, Shalini Nair, Ian H Cheeseman, Cherise Rohr-Allegrini, Sittaporn Singlam, Francois Nosten, and Tim J C Anderson. (2012) 2012. “Close Kinship Within Multiple-Genotype Malaria Parasite Infections.”. Proceedings. Biological Sciences 279 (1738): 2589-98. https://doi.org/10.1098/rspb.2012.0113.

Malaria infections containing multiple parasite genotypes are ubiquitous in nature, and play a central role in models of recombination, intra-host dynamics, virulence, sex ratio, immunity and drug resistance evolution in Plasmodium. While these multiple infections (MIs) are often assumed to result from superinfection (bites from multiple infected mosquitoes), we know remarkably little about their composition or generation. We isolated 336 parasite clones from eight patients from Malawi (high transmission) and six from Thailand (low transmission) by dilution cloning. These were genotyped using 384 single-nucleotide polymorphisms, revealing 22 independent haplotypes in Malawi (2-6 per MI) and 15 in Thailand (2-5 per MI). Surprisingly, all six patients from Thailand and six of eight from Malawi contained related haplotypes, and haplotypes were more similar within- than between-infections. These results argue against a simple superinfection model. Instead, the observed kinship patterns may be explained by inoculation of multiple related haploid sporozoites from single mosquito bites, by immune suppression of parasite subpopulations within infections, and serial transmission of related parasites between people. That relatedness is maintained in endemic areas in the face of repeated bites from infected mosquitoes has profound implications for understanding malaria transmission, immunity and intra-host dynamics of co-infecting parasite genotypes.

Cheeseman, Ian H, Becky A Miller, Shalini Nair, Standwell Nkhoma, Asako Tan, John C Tan, Salma Al Saai, et al. (2012) 2012. “A Major Genome Region Underlying Artemisinin Resistance in Malaria.”. Science (New York, N.Y.) 336 (6077): 79-82. https://doi.org/10.1126/science.1215966.

Evolving resistance to artemisinin-based compounds threatens to derail attempts to control malaria. Resistance has been confirmed in western Cambodia and has recently emerged in western Thailand, but is absent from neighboring Laos. Artemisinin resistance results in reduced parasite clearance rates (CRs) after treatment. We used a two-phase strategy to identify genome region(s) underlying this ongoing selective event. Geographical differentiation and haplotype structure at 6969 polymorphic single-nucleotide polymorphisms (SNPs) in 91 parasites from Cambodia, Thailand, and Laos identified 33 genome regions under strong selection. We screened SNPs and microsatellites within these regions in 715 parasites from Thailand, identifying a selective sweep on chromosome 13 that shows strong association (P = 10(-6) to 10(-12)) with slow CRs, illustrating the efficacy of targeted association for identifying the genetic basis of adaptive traits.

2011

Tan, John C, Becky A Miller, Asako Tan, Jigar J Patel, Ian H Cheeseman, Tim J C Anderson, Magnus Manske, Gareth Maslen, Dominic P Kwiatkowski, and Michael T Ferdig. (2011) 2011. “An Optimized Microarray Platform for Assaying Genomic Variation in Plasmodium Falciparum Field Populations.”. Genome Biology 12 (4): R35. https://doi.org/10.1186/gb-2011-12-4-r35.

We present an optimized probe design for copy number variation (CNV) and SNP genotyping in the Plasmodium falciparum genome. We demonstrate that variable length and isothermal probes are superior to static length probes. We show that sample preparation and hybridization conditions mitigate the effects of host DNA contamination in field samples. The microarray and workflow presented can be used to identify CNVs and SNPs with 95% accuracy in a single hybridization, in field samples containing up to 92% human DNA contamination.

2009

Cheeseman, Ian H, Natalia Gomez-Escobar, Celine K Carret, Alasdair Ivens, Lindsay B Stewart, Kevin K A Tetteh, and David J Conway. (2009) 2009. “Gene Copy Number Variation Throughout the Plasmodium Falciparum Genome.”. BMC Genomics 10: 353. https://doi.org/10.1186/1471-2164-10-353.

BACKGROUND: Gene copy number variation (CNV) is responsible for several important phenotypes of the malaria parasite Plasmodium falciparum, including drug resistance, loss of infected erythrocyte cytoadherence and alteration of receptor usage for erythrocyte invasion. Despite the known effects of CNV, little is known about its extent throughout the genome.

RESULTS: We performed a whole-genome survey of CNV genes in P. falciparum using comparative genome hybridisation of a diverse set of 16 laboratory culture-adapted isolates to a custom designed high density Affymetrix GeneChip array. Overall, 186 genes showed hybridisation signals consistent with deletion or amplification in one or more isolate. There is a strong association of CNV with gene length, genomic location, and low orthology to genes in other Plasmodium species. Sub-telomeric regions of all chromosomes are strongly associated with CNV genes independent from members of previously described multigene families. However, approximately 40% of CNV genes were located in more central regions of the chromosomes. Among the previously undescribed CNV genes, several that are of potential phenotypic relevance are identified.

CONCLUSION: CNV represents a major form of genetic variation within the P. falciparum genome; the distribution of gene features indicates the involvement of highly non-random mutational and selective processes. Additional studies should be directed at examining CNV in natural parasite populations to extend conclusions to clinical settings.

Walther, Michael, David Jeffries, Olivia C Finney, Madi Njie, Augustine Ebonyi, Susanne Deininger, Emma Lawrence, et al. (2009) 2009. “Distinct Roles for FOXP3 and FOXP3 CD4 T Cells in Regulating Cellular Immunity to Uncomplicated and Severe Plasmodium Falciparum Malaria.”. PLoS Pathogens 5 (4): e1000364. https://doi.org/10.1371/journal.ppat.1000364.

Failure to establish an appropriate balance between pro- and anti-inflammatory immune responses is believed to contribute to pathogenesis of severe malaria. To determine whether this balance is maintained by classical regulatory T cells (CD4(+) FOXP3(+) CD127(-/low); Tregs) we compared cellular responses between Gambian children (n = 124) with severe Plasmodium falciparum malaria or uncomplicated malaria infections. Although no significant differences in Treg numbers or function were observed between the groups, Treg activity during acute disease was inversely correlated with malaria-specific memory responses detectable 28 days later. Thus, while Tregs may not regulate acute malarial inflammation, they may limit memory responses to levels that subsequently facilitate parasite clearance without causing immunopathology. Importantly, we identified a population of FOXP3(-), CD45RO(+) CD4(+) T cells which coproduce IL-10 and IFN-gamma. These cells are more prevalent in children with uncomplicated malaria than in those with severe disease, suggesting that they may be the regulators of acute malarial inflammation.

2008

Wilkinson, Shane R, Martin C Taylor, David Horn, John M Kelly, and Ian Cheeseman. (2008) 2008. “A Mechanism for Cross-Resistance to Nifurtimox and Benznidazole in Trypanosomes.”. Proceedings of the National Academy of Sciences of the United States of America 105 (13): 5022-7. https://doi.org/10.1073/pnas.0711014105.

Nifurtimox and benznidazole are the front-line drugs used to treat Chagas disease, the most important parasitic infection in the Americas. These agents function as prodrugs and must be activated within the parasite to have trypanocidal effects. Despite >40 years of research, the mechanism(s) of action and resistance have remained elusive. Here, we report that in trypanosomes, both drugs are activated by a NADH-dependent, mitochondrially localized, bacterial-like, type I nitroreductase (NTR), and that down-regulation of this explains how resistance may emerge. Loss of a single copy of this gene in Trypanosoma cruzi, either through in vitro drug selection or by targeted gene deletion, is sufficient to cause significant cross-resistance to a wide range of nitroheterocyclic drugs. In Trypanosoma brucei, loss of a single NTR allele confers similar cross-resistance without affecting growth rate or the ability to establish an infection. This potential for drug resistance by a simple mechanism has important implications, because nifurtimox is currently undergoing phase III clinical trials against African trypanosomiasis.

  • Dia, Aliou, and Ian H Cheeseman. (2021) 2021. “Single-Cell Genome Sequencing of Protozoan Parasites.”. Trends in Parasitology 37 (9): 803-14. https://doi.org/10.1016/j.pt.2021.05.013.

    Despite considerable genetic variation within hosts, most parasite genome sequencing studies focus on bulk samples composed of millions of cells. Analysis of bulk samples is biased toward the dominant genotype, concealing cell-to-cell variation and rare variants. To tackle this, single-cell sequencing approaches have been developed and tailored to specific host-parasite systems. These are allowing the genetic diversity and kinship in complex parasite populations to be deciphered and for de novo genetic variation to be captured. Here, we outline the methodologies being used for single-cell sequencing of parasitic protozoans, such as Plasmodium and Leishmania spp., and how these tools are being applied to understand parasite biology.

    See also: Infectious Disease