Research

Malaria Research Projects

Malaria parasites in red blood cells (Fluorescent microscopy) Artemisinin resistance: Our laboratory first determined that the “slow parasite clearance” observed in Cambodia was due to parasite genetics. We showed that the genes responsible for slow clearance were increasing in frequency along the Thai-Myanmar border and subsequently located the genomic region responsible for much of this variation. Following the identification of Kelch13 in this genomic region, we have documented the dynamics of soft selective sweeps at this locus along the Thailand-Myanmar border.

Representative publications from the lab:

Graph showing a selective sweep purging genetic variation from chr 4 surrounding dihydrofolate reductase driven by pyrimethamine treatment in SE Asia. The same resistance alleles subsequently spread across sub-Saharan Africa (adapted from Nair et al. Mol Biol Evol 2003) Dynamics of drug resistance evolution: Drug resistance evolution provides a valuable model for understanding adaptation in nature and has significant implications for public health policy. Our group demonstrated that high-level pyrimethamine resistance originated from a single source in Southeast Asia, with a selective sweep around the dihydrofolate reductase (dhfr) gene leading to the loss of genetic variation on chromosome 4. The subsequent transfer of pyrimethamine resistance alleles from Asia to Africa resulted in a significant decrease in the efficacy of antifolate drugs. We have also investigated the role of copy number variation (CNV) in this resistance phenotype, revealing multiple independent origins of copy number amplification in an ABC transporter and demonstrating adaptive changes in CNV during the evolution of antifolate resistance.

Representative publications from the lab:

Genetic structure of parasite populations: Our initial work on malaria developed and utilized microsatellite markers to characterize Plasmodium falciparum populations, revealing dramatic differences in population structure that correlate closely with transmission intensity. We recently observed similar changes in genetic structure within a single population where transmission was declining. Our lab has focused on the genetic composition of multiple-clone infections, demonstrating that these infections consist of related parasite haplotypes, which contradicts simpler models of parasite superinfection. This finding was recently confirmed using FACS-based single-cell genomics methods developed in our laboratory.

Representative publications from the Lab:

Structural variation in parasite DNA: Large-scale deletions, copy number variations (CNVs), insertions, and rearrangements are common in pathogen genomes exhibit high mutation rates, and may underpin rapid adaptation to drugs and other selective pressures. However, structural variants are more challenging to score than SNPs or Indels and have received less attention. We have investigated the role of CNVs in drug resistance evolution and compensatory mutations, as well as the population genomics of CNVs in Plasmodium populations worldwide. We are also using Nanopore long-read sequencing to provide parent-specific references for P. falciparum genetic crosses conducted in our lab. We anticipate that this approach will enhance our ability to identify segregating variants across complex genomic regions that are difficult to access using short-read Illumina sequencing alone.

Representative publications from the Lab:

Shoklo camp on the Thailand-Myanmar border Genomic epidemiology during malaria elimination in Myanmar: We are currently generating and using genomic data from P. falciparum parasites collected from infected patients on the Thai-Myanmar border to better understand malaria transmission during intensive malaria control efforts. This work is conducted in collaboration with Dr. François Nosten (Shoklo Malaria Research Unit) and Dr. Daniel Parker.

 

 

Through a P01 NIH grant, we have been collaborating with Dr. Ashley Vaughan (Seattle Children’s Hospital), Dr. Mike Ferdig (University of Notre Dame), and Dr. Ian Cheeseman (Texas Biomedical Research Institute) to utilize humanized mice for conducting genetic crosses in P. falciparum. This collaboration has enabled us to generate crosses with large numbers of progeny, providing excellent statistical power to localize genes underlying important parasite phenotypic traits. Specifically, we have focused on developing efficient bulk-segregant approaches for analyzing genetic crosses, which do not require laborious cloning and phenotyping of individual progeny. We have applied these methods to identify genomic regions associated with parasite fitness and nutrient acquisition/metabolism.

Representative publications from the lab:

Scheme of CRISPR editing in malaria parasites Fitness costs, compensation and epistasis: Drug resistance genes often interfere with key metabolic processes, leading to significant fitness costs for the parasites. We anticipate that compensatory changes will evolve to restore fitness in drug-resistant parasites. Different genes involved in drug resistance evolution may interact in additive or epistatic ways to influence both drug resistance and parasite fitness. We are employing various approaches to identify and understand fitness costs and compensatory evolution in malaria, and to determine whether gene interactions are additive or epistatic in shaping outcome phenotypes. Specifically, we are using experimental evolution, longitudinal population genomics surveys, and CRISPR/Cas9 techniques to gain insights into the roles of fitness, compensation, and epistasis in drug resistance evolution.

Representative publications from the lab:

Deletions of Histidine Rich proteins and diagnostic evasion in malaria parasites: Histidine-rich proteins are produced in large quantities by P. falciparum and form the basis of rapid diagnostic tests for malaria. However, some parasites carry deletions in the genes encoding these proteins. We are investigating the costs associated with these gene deletions and the compensatory mutations that may emerge to counteract the resulting fitness costs in the parasites.

Representative publications from the lab:

Schistosome Research Projects

Cover of Science Translational Medicine showing a schistosome male worms Our lab pioneers the use of laboratory genetic crosses combined with linkage analysis, bulk-segregant analysis, and genome-wide association studies to map the genetic bases of oxamniquine and praziquantel resistance in schistosome parasites. Using these genetic and genomic approaches, we have deciphered the mechanism of action of oxamniquine, a drug used in South America to treat infections with Schistosoma mansoni.

Praziquantel (PZQ) is currently the only drug available to treat schistosomiasis, but resistance could occur in the field. Using a genome-wide association study (GWAS) with a lab population of parasites polymorphic for PZQ resistance, coupled with a new high-throughput phenotyping method based on worm metabolism, we have pinpointed a genomic region involved in PZQ response. This discovery allows us to identify a candidate gene (TRPM.PZQ) involved in PZQ's mode of action, which was previously unknown. Deciphering the PZQ response gene(s) will enable us to: (i) identify genetic markers linked to drug resistance or reduced efficacy, and (ii) develop molecular surveillance approaches to detect drug-resistant parasites in the field, assisting current mass drug administration programs in Afri Schistosomiasis is an occupational hazard for fishing communities on the shores of lake Victoria ca and adapting treatments.

We are currently collaborating with Dr. Eric Ndombi and his team in Kisumu, Western Kenya, Dr. Yves-Nathan Tian-Bi in Côte d’Ivoire, Dr. Kader Kondé in Guinea, Dr. Steffi Knopp, Dr. Mohammed Said, and Dr. Humphrey Mazigo in Zanzibar Tanzania to investigate the genetic diversity of the TRPM.PZQ gene in field-collected samples of S. mansoni and S. haematobium. Functional validation of any mutations found in the field will be analyzed for PZQ resistance/susceptibility phenotypes through our collaboration with Prof. Jonathan Marchant at the Medical College of Wisconsin.

Representative publications from the Lab:

Biomphalaria alexandrina snails, pigmented and coming from Egypt. These snails are vector of Schistosoma mansoni. Host-specificity: Host-pathogen compatibility is determined by both host and parasite genes. There are multiple models of how this evolves (gene-for-gene coevolution and gene matching models), but there are few examples where this is understood at the molecular level. We are using genetic crosses and bulk-segregant approaches to determine the Schistosome parasite genes involved in compatibility with their snail hosts. On the other side of the interaction, the Blouin lab is using similar genetic approaches to identify the snail genes involved in compatibility and resistance to schistosome parasites. We hope to understand the molecules involved in these compatibility and resistance mechanisms in both the snail and the schistosome parasite.

Schistosoma mansoni cercariae shed from an infected Biomphalaria snail Cercarial production and chronobiology: We have characterized striking differences in virulence, transmission, and parasite growth dynamics between two Schistosoma mansoni populations. One population of parasites is particularly virulent for snail and mammal hosts, linked to significant production of sporocysts and cercariae in the snail host and eggs in the mammal host, while the other exhibits a low virulence phenotype. By conducting genetic crosses between parasites from these two populations, we have revealed that cercarial production involves multiple genes. Identifying the causative genes will require a better understanding of sporocyst biology and the different cell types that constitute them.

Cercarial production is not continuous but rather rhythmic and dependent on the time of day. For example, some schistosome parasites shed cercariae during daylight, while others shed cercariae at night. Using genetic crosses and classical linkage mapping between populations of diurnal and nocturnal S. mansoni, we aim to decipher the genetic basis of these chronobiological adaptive traits. Our work on cercarial chronobiology is done in collaboration with Dr. Hélène Moné and Dr. Gabriel Mouahid  from the University of Perpignan in France.

Understanding how parasites adapt and develop within their hosts is crucial for identifying new therapeutic targets, leading to better control of schistosome parasites and, eventually, the schistosomiasis disease.

Representative publications from the Lab:

Single-cell transcriptomics of Schistosome sporocysts: Our goal is to develop an atlas of sporocyst cell types and to identify differences in cell composition and gene expression of these cell types that could be linked to cercarial production. This atlas will be a valuable resource for the schistosome research community and could be used to develop cell-type markers for spatial transcriptomic approaches.

Schistosome invasion of the Americas: Using whole genome amplification and sequencing of single miracidia larvae from infected patients preserved on FTA cards, we examined the genomic impact of Schistosoma mansoni colonization in South America during the transatlantic slave trade. We found a significant reduction in genetic diversity and slower decay in linkage disequilibrium (LD) in parasites from East to West Africa. Despite this, nuclear diversity and LD in West Africa and Brazil were similar, indicating minimal bottlenecks and barriers to colonization. Our data imply that unsampled populations from central Africa, including contributions from Niger, were likely the main sources of Brazilian S. mansoni. The lack of a bottleneck suggests that this invasion was likely facilitated by pre-adapted parasites that established themselves relatively easily in the Americas.

Representative publications from the Lab:

Schistosome hybridization: Schistosoma haemat obium, which causes urinary schistosomiasis in humans, and the related livestock parasite Schistosoma bovis can interbreed in the laboratory and produce fertile offspring. Previous studies have suggested ongoing hybridization between these species and others in the S. haematobium clade, though they used limited genetic markers. Using whole genome amplification and exome/genome sequencing of single S. haematobium miracidia larvae preserved on FTA cards from infected patients in West and East Africa, we found no evidence of contemporary hybridization between S. bovis and S. haematobium. However, all S. haematobium genomes from West Africa showed hybrid ancestry, with 3.3-8.2% of their nuclear genomes derived from S. bovis, indicating an ancient introgression event. We are now combining population genomics and laboratory genetic crosses to further investigate the genomic consequences, frequency, and phenotypic effects of this introgression.

Representative publications from the Lab:

Long read sequencing to maximize the utility of parasite genetic crosses: Schistosome genomes are rich in transposable elements (TEs) and structural rearrangements, which are challenging to resolve with Illumina short-read sequencing alone. We are using Nanopore long-read sequencing to generate parent-specific reference sequences for the schistosome genetic crosses conducted in our lab. We anticipate that this approach will enhance our ability to align reads across the entire genome and identify TE insertions and structural variants that may be driving quantitative trait loci (QTLs) linked to several phenotypes of interest that we are investigating.

Dr. Frédéric Chevalier and Dr. Winka Le Clec’h, staff scientists in the Anderson lab, are co-leading a schistosome-snail-host-microbiome project. They investigate the diversity and abundance of microorganisms within the hemolymph (i.e., blood) and organs of Biomphalaria snails. They have highlighted significant differences in microbiome composition at the level of individual snails, snail populations, and species. They have also established a snail organs microbiome atlas and are currently investigating the impact of schistosome infections on the snail microbiome with NIH R21 support. Understanding the role of the microbiome in the snail-parasite interaction can lead to better control of parasite populations, especially if specific bacteria help snails resist infection.

Representative publications from the Lab:

Main technologies and methods used:

Malaria parasites in red blood cells Parasite lifecycles: In our laboratory, we actively maintain the full Schistosome worms - male and female paired together lifecycle of several populations of schistosome parasites (S. mansoni and S. haematobium) using our snail colonies and rodents (hamsters or mice). We currently have inbred and outbred snail populations of Biomphalaria glabrata from Brazil, B. alexandrina from Egypt, B. sudanica from Kenya, Bulinus truncatus, and Bulinus globosus from Kenya. Additionally, we have a diverse collection of cryopreserved Plasmodium falciparum parasites (from various African and Southeast Asian countries), which we can propagate asexually using human blood.

Molecular biology and cell biology: In our laboratory, we use a wide variety of molecular biology and cell biology techniques, which we have developed and/or adapted for our non-model organisms of interest: Plasmodium falciparum and Schistosoma mansoni. These techniques include:

  • Library preparation for high-throughput sequencing (Whole Genome sequencing, Exome capture, RNAseq, MiSeq, Nanopore Long-read sequencing).
  • Targeted-capture array
  • Whole genome amplification (from FTA card preserved samples or gDNA)
  • PCR / Nested PCR / quantitative PCR / RT-PCR and qRT-PCR / PCR-RFLP   
  • In-vitro culture and assay for P. falciparum and Schistosome worms    
  • Single-cell RNAseq for Malaria parasites and Schistosome sporocysts
  • CRISPR gene editing
  • Phenoloxidase activity dosage in snail blood
  • Drug treatment assay (for both Malaria and Schistosome worms)

Data analysis: All data generated in our lab are analyzed in-house using custom pipelines (Conda environments, etc.) and scripts (Bash, R, Python, etc.) on the Texas Biomed High-Performance Computing Cluster. These analyses include:

 

  • WGS, Exome, Microbiome, RNAseq, scRNAseq pipelines for Malaria and Schistosome parasites
  • Quantitative Trait Loci (QTL) analysis
  • Bulk-segregant analysis
  • GWAS analysis
  • Phylogenetic analysis
  • Population genomics
  • Admixture analysis
  • Statistical analysis using R (survival analysis, multivariate analysis etc.)