Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites

<p>Abstract</p> <p>Background</p> <p>Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine <it>in vivo </it>experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, <it>Plasmodium chabaudi</it>. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, <it>P. falciparum</it>.</p> <p>Results</p> <p>A lineage of isogenic <it>in vivo </it>drug-selected mutant <it>P. chabaudi </it>parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an <it>in vivo </it>artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina^®Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme.</p> <p>Conclusions</p> <p>This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.</p

Royal Society Discussion Meeting: Utilising the Genome Sequence of Parasitic Protozoa

Protozoan parasites cause some of the world’s most important diseases. Genome sequencing information is rapidly being acquired and combined with new developments in functional genome analysis to transform our understanding of parasites, and to enable new approaches to combating the diseases they cause

Environmental constraints on the physiology of transgenic mosquitoes

Insects have exploited and responded to their environment in a plethora of ways. Environmental changes are used to trigger short- and long-term physiological events and environmental stresses have resulted in evolution of gene families and resistance genes. This highly evolved, tight interaction between organism and environment will be altered in transgenic mosquitoes, and this paper reviews some potential considerations concerning the physiology of transgenic mosquitoes upon release. This papers examines a few of the recent discoveries in Plasmodium-mosquito interactions and discusses the impact upon them of the transgenic-mosquito approach. A complex interplay between vector and parasite occurs during transmission, including the exploitation of xanthurenic acid for triggering exflagellation, the induction of a mosquito immune response and its evasion by the invading ookinete, and the ability of the parasite to establish infections when major genes are knocked out. Such functional redundancy in parasite genes is also demonstrable in the immune and detoxification systems of insects. Consequently, where genes can substitute for one another in a given physiological process, there is potentially significant environmental pressure for differential gene expression. In the transgenic context, such compensatory regulation could work to down-regulate and/or select against a transgene. Conversely, additional environmental triggers could be exploited to select positively for a transgenic mosquito. There is potential for heterogeneity at each stage of the transgenic release strategy, and addressing this will be important if such approaches are not to be scuttled by unforeseen factors that could reduce expression of and selection for the beneficial transgene

Integration and mining of malaria molecular, functional and pharmacological data: how far are we from a chemogenomic knowledge space?

The organization and mining of malaria genomic and post-genomic data is highly motivated by the necessity to predict and characterize new biological targets and new drugs. Biological targets are sought in a biological space designed from the genomic data from Plasmodium falciparum, but using also the millions of genomic data from other species. Drug candidates are sought in a chemical space containing the millions of small molecules stored in public and private chemolibraries. Data management should therefore be as reliable and versatile as possible. In this context, we examined five aspects of the organization and mining of malaria genomic and post-genomic data: 1) the comparison of protein sequences including compositionally atypical malaria sequences, 2) the high throughput reconstruction of molecular phylogenies, 3) the representation of biological processes particularly metabolic pathways, 4) the versatile methods to integrate genomic data, biological representations and functional profiling obtained from X-omic experiments after drug treatments and 5) the determination and prediction of protein structures and their molecular docking with drug candidate structures. Progresses toward a grid-enabled chemogenomic knowledge space are discussed.Comment: 43 pages, 4 figures, to appear in Malaria Journa

First report of natural Wolbachia infection in the malaria mosquito Anopheles arabiensis in Tanzania

Background: Natural infections of the endosymbiont bacteria Wolbachia have recently been discovered in populations of the malaria mosquito Anopheles gambiae (s.l.) in Burkina Faso and Mali, West Africa. This Anopheles specific strain wAnga limits the malaria parasite Plasmodium falciparum infections in the mosquito, thus it offers novel opportunities for malaria control. Results: We investigated Wolbachia presence in Anopheles arabiensis and Anopheles funestus, which are the two main malaria vectors in the Kilombero Valley, a malaria endemic region in south-eastern Tanzania. We found 3.1% (n = 65) and 7.5% (n = 147) wAnga infection prevalence in An. arabiensis in mosquitoes collected in 2014 and 2016, respectively, while no infection was detected in An. funestus (n = 41). Phylogenetic analysis suggests that at least two distinct strains of wAnga were detected, both belonging to Wolbachia supergroup A and B. Conclusions: To our knowledge, this is the first confirmation of natural Wolbachia in malaria vectors in Tanzania, which opens novel questions on the ecological and genetic basis of its persistence and pathogen transmission in the vector hosts. Understanding the basis of interactions between Wolbachia, Anopheles mosquitoes and malaria parasites is crucial for investigation of its potential application as a biocontrol strategy to reduce malaria transmission, and assessment of how natural wAnga infections influence pathogen transmission in different ecological settings

Proteomics of Anopheles gambiae

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Perspectives for Virulence Management: Relating Theory to Experiment

This paper reviews our current knowledge about the evolution of virulence in pathogen-host systems, with an emphasis on the interface between the theoretical and experimental literature. After giving a methodically oriented overview of the field, stressing restrictions and caveats, the paper attempts to summarize the main results on virulence evolution gleaned from the literature. From that perspective the authors identify what they see as gaps in our current knowledge that need to be filled to transform the study of virulence evolution and management into a mature science

Transcriptional Regulation and Epigenetic Mechanisms Underlying Host-Parasite Interactions in Human Malaria

Human malaria is one of the most important infectious diseases and a major cause of death and poverty worldwide. It is caused by protozoan parasites of the genus Plasmodium that are transmitted by the bites of mosquitoes of the genus Anopheles. The parasites Plasmodium falciparum and the mosquitoes Anopheles gambiae are the leading figures of this global burden, which disproportionally affects Africa and children under the age of five. To fulfill development and to achieve adaptation to changing environments in the human and mosquito hosts, Plasmodium parasites are capable of drastic transcriptional switches. The Anopheles mosquitoes are the main vectors for human malaria, and they can display phenotypic variability in life history traits, including vector competence or responses against Plasmodium. Yet, the transcriptional regulation underlying host parasite interactions in human malaria, particularly based on epigenetic mechanisms and regarding the life cycle in the mosquito, remain almost completely unknown. In this doctoral thesis, we have applied multi-omic approaches and bioinformatic analyses to investigate the regulatory genome of both P. falciparum and A. gambiae mosquitoes, associated with the Plasmodium development and interactions within hosts, and with the responses of Anopheles mosquitoes to the parasitic infection. We have integrated genomic, epigenomic and transcriptomic approach to unveil relevant cis-regulatory elements and to assay the relationship between gene expression levels and chromatin-related mechanisms, such as histone marks or chromatin accessibility levels. We applied different techniques to these organisms, integrating RNA-seq, ChIP-seq and ATAC-seq for the first time. We reported the positive correlation between transcription and chromatin accessibility by ATAC-seq or active histone marks by ChIP-seq. We also identified thousands of active regulatory sequences, including enhancer candidates, that appeared to be linked to Plasmodium developmental transitions or clonally variant gene expression within humans, or that in the case of mosquitoes seemed to be specific to tissues or Plasmodium infection status. Ultimately, these allowed us to predict cognate transcription factors. Altogether, we provide evidence for genome-wide mechanisms and regulatory regions that may be involved in the dynamic transcriptional regulation underlying host-parasite interactions between malaria parasites and the human and mosquito hosts. This is much required in the context of current efforts against malaria, to inform existing and new mosquito-control and anti-malaria strategies