Genomics and spatial surveillance of Chagas disease and American visceral leishmaniasis

Schwabl, Philipp (2020) Genomics and spatial surveillance of Chagas disease and American visceral leishmaniasis. PhD thesis, University of Glasgow.

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Abstract

The Trypanosomatidae are a family of parasitic protozoa that infect various animals and plants. Several species within the Trypanosoma and Leishmania genera also pose a major threat to human health. Among these are Trypanosoma cruzi and Leishmania infantum, aetiological agents of the highly debilitating and often deadly vector-borne zoonoses Chagas disease and American visceral leishmaniasis. Current treatment options are far from safe, only partially effective and rarely available in the impoverished regions of Latin America where these ‘neglected tropical diseases’ prevail. Wider-reaching, sustainable protection against T. cruzi and L. infantum might best be achieved by intercepting key routes of zoonotic transmission, but this prophylactic approach requires a better understanding of how these parasites disperse and evolve at various spatiotemporal scales.
This dissertation addresses key questions around trypanosomatid parasite biology and spatial epidemiology based on high-resolution, geo-referenced DNA sequence datasets constructed from disease foci throughout Latin America:
Which forms of genetic exchange occur in T. cruzi, and are exchange events frequent enough to significantly alter the distribution of important epidemiological traits? How do demographic histories, for example, the recent invasive expansion of L. infantum into the Americas, impact parasite population structure, and do structural changes pose a threat to public health? Can environmental variables predict parasite dispersal patterns at the landscape scale?
Following the first chapter’s review of population genetic and genomic approaches in the study of trypanosomatid diseases in Latin America, Chapter 2 describes how reproductive polymorphism segregates T. cruzi populations in southern Ecuador. The study is the first to clearly demonstrate meiotic sex in this species, for decades thought to exchange genetic material only very rarely, and only by non-Mendelian means. T. cruzi subpopulations from the Ecuadorian study site exhibit all major hallmarks of sexual reproduction, including genome-wide Hardy-Weinberg allele frequencies, rapid decay of linkage disequilibrium with map distance and genealogies that fluctuate among chromosomes. The presence of sex promotes the transfer and transformation of genotypes underlying important epidemiological traits, posing great challenges to disease surveillance and the development of diagnostics and drugs.
Chapter 3 demonstrates that mating events are also pivotal to L. infantum population structure in Brazil, where introduction bottlenecks have led to striking genetic discontinuities between sympatric strains. Genetic hybridization occurs genome-wide, including at a recently identified ‘miltefosine sensitivity locus’ that appears to be deleted from the majority of Brazilian L. infantum genomes. The study combines an array of genomic and phenotypic analyses to determine whether rapid population expansion or strong purifying selection has driven this prominent > 12 kb deletion to high abundance across Brazil. Results expose deletion size differences that covary with phylogenetic structure and suggest that deletion-carrying strains do not form a private monophyletic clade. These observations are inconsistent with the hypothesis that the deletion genotype rose to high prevalence simply as the result of a founder effect. Enzymatic assays show that loss of ecto-3’-nucleotidase gene function within the deleted locus is coupled to increased ecto-ATPase activity, raising the possibility that alternative metabolic strategies enhance L. infantum fitness in its introduced range. The study also uses demographic simulation modelling to determine whether L. infantum populations in the Americas have expanded from just one or multiple introduction events. Comparison of observed vs. simulated summary statistics using random forests suggests a single introduction from the Old World, but better spatial sampling coverage is required to rule out other demographic scenarios in a pattern-process modelling approach. Further sampling is also necessary to substantiate signs of convergent selection introduced above.
Chapter 4 therefore develops a ‘genome-wide locus sequence typing’ (GLST) tool to summarize parasite genetic polymorphism at a fraction of genomic sequencing cost. Applied directly to the infection source (e.g., vector or host tissue), the method also avoids bias from cell purification and culturing steps typically involved prior to sequencing of trypanosomatid and other obligate parasite genomes. GLST scans genomic pilot data for hundreds of polymorphic sequence fragments whose thermodynamic properties permit simultaneous PCR amplification in a single reaction tube. For proof of principle, GLST is applied to metagenomic DNA extracts from various Chagas disease vector species collected in Colombia, Venezuela, and Ecuador. Epimastigote DNA from several T. cruzi reference clones is also analyzed. The method distinguishes 387 single-nucleotide polymorphisms (SNPs) in T. cruzi sub-lineage TcI and an additional 393 SNPs in non-TcI clones. Genetic distances calculated from these SNPs correlate with geographic distances among samples but also distinguish parasites from triatomines collected at common collection sites. The method thereby appears suitable for agent-based spatio-genetic (simulation) analyses left wanted by Chapter 3 – and further formulated in Chapter 5.
The potential to survey parasite genetic diversity abundantly across landscapes compels deeper, more systematic exploration of how environmental variables influence the spread of disease. As environmental context is only marginally considered in the population genetic analyses of Chapters 2 – 4, Chapter 5 proposes a new, spatially explicit modelling framework to predict vector-borne parasite gene flow through heterogeneous environment. In this framework, remotely sensed environmental raster values are re-coded and merged into a composite ‘resistance surface’ that summarizes hypothesized effects of landscape features on parasite transmission among vectors and hosts. Parasite population genetic differentiation is then simulated on this surface and fitted to observed diversity patterns in order to evaluate original hypotheses on how environmental variables modulate parasite gene flow. The chapter thereby makes a maiden step from standard population genetic to ‘landscape genomic’ approaches in understanding the ecology and evolution of vector-borne disease.
In summary, this dissertation first demonstrates the power of population genetics and genomics to understand fundamental biological properties of important protist parasites, then identifies areas where analytical tools are missing and creates new technical and conceptual frameworks to help fill these gaps. The general discussion (Chapter 6) also outlines several follow-up projects on the key finding of meiotic genetic signatures in T. cruzi. Exploiting recently developed T. cruzi genome-editing systems for the detection of meiotic gene expression and heterozygosis will help understand why and in which life cycle stage some parasite populations use sex and others do not. Long-read sequencing of parental and recombinant genomes will help understand the extent to which sex is diversifying T. cruzi phenotypes, especially virulence and drug resistance properties conferred by surface molecules with repetitive genetic bases intractable to short-read analysis. Chapter 6 also provides follow-up plans for all other research chapters. Emphasis is placed on advancing the complementarity, transferability and public health benefit of the many different methods and concepts employed in this work.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > Q Science (General)
Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Biodiversity Animal Health and Comparative Medicine
Supervisor's Name: Llewellyn, Dr. Martin S.
Date of Award: 2020
Depositing User: Dr. Philipp Schwabl
Unique ID: glathesis:2020-81448
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 16 Jun 2020 06:26
Last Modified: 16 Jun 2020 06:35
URI: http://theses.gla.ac.uk/id/eprint/81448

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