Adaptive variation in sexual and asexual reproduction of endemic malaria parasites

Malaria caused by Plasmodium falciparum is responsible for approximately half a million deaths and hundreds of millions of clinical cases annually. In studies of multiple endemic populations, we have recently uncovered strong evidence of local adaptation on a locus regulating parasite sexual development, and have found remarkable variation in asexual multiplication rates of this parasite in endemic areas.
This proposal is to undertake a quantitative analysis of sexual and asexual reproduction in malaria parasites from endemic populations, and test a hypothesis of transcriptional control and selection. This will enable evaluation of cellular markers for identifying alternatively committed parasites in the generation cycle before morphological differentiation, which may be targets for interventions to reduce transmission or disease.
The study aims to answer the following questions:

1) What are distributions of naturally occurring switch rates to sexual differentiation of Plasmodium falciparum?
We will measure and compare rates of commitment to sexual stage differentiation per asexual generation cycle in parasites isolated from clinical infections in areas with high levels of transmission, moderate seasonal transmission, and extremely low transmission. Basal per-generation rates will be measured as well as responses to induction of gametocytogenesis by transfer to growth medium lacking lysophosphatidylcholine.

2) What are the principal determinants of asexual multiplication rate variation?
We will test for correlations between sexual commitment rates and asexual multiplication rates within each of the three panels of clinical isolates studied in the previous section. In addition, in a subset of isolates components of asexual multiplication will be quantified by measuring numbers of merozoites per mature schizont, merozoite invasion efficiency, use of alternative receptors for invasion, and cell cycle duration. The overall variance in each of the measured parameters and their associations with asexual multiplication rate will be tested by partial correlation analyses.

3) How do the alternative major haplotypes of the gdv1 3'-intergenic region affect transcription and sexual commitment?
The locus under strongest local differentiation among populations in West Africa is gdv1 and its 3'-intergenic region, which silenced by antisense RNA in cultured parasites. The levels of 3'-intergenic and antisense long non-coding RNA transcripts, in comparison with the sense transcripts of gdv1, will be analysed in clinical isolates before and after induction to switch to gametocytes. The basal strand-specific transcript levels, and the alteration in strand-specific transcripts in response to the induction, will be tested for differences between the two major gdv1 3'-intergenic allelic types. The variation in conversion rate among the lines will be tested for correlation with changes in strand-specific transcripts in response to induction. Complementary to this association test, allelic replacement genetic modifications will be performed to determine the regulatory effects of different parts of the intergenic sequence.

Further areas of investigation that would capitalise on the understanding and tools gained from the above approaches include some work that may be achievable within this project if time allows, and some that would require separate funding. For example, we would test whether the variably-expressed merozoite surface protein MSPDBL2 is exclusive to parasites committed to become sexually differentiated in the cycle generation. If so, cell sorting will enable identification of other merozoite proteins associated with commitment to sexual development, potential targets for transmission-blocking.

This proposal is to undertake a quantitative analysis of sexual and asexual reproduction in malaria parasites from endemic populations, and test a hypothesis of transcriptional control and selection.
It addresses the following questions:

1) What are distributions of naturally occurring switch rates to sexual differentiation of Plasmodium falciparum?
Rates of commitment to sexual stage differentiation per asexual generation cycle will be measured in parasites isolated from clinical infections in areas with high levels of transmission (in Ghana), moderate seasonal transmission (in The Gambia), and extremely low transmission (in Malaysia). Induction of gametocytogenesis will be performed by transfer to growth medium lacking lysophosphatidylcholine.

2) What are the principal determinants of asexual multiplication rate variation?
We will test for correlations between sexual commitment rates and asexual multiplication rates within each of the panels of clinical isolates studied in the previous section. Components of the asexual multiplication cycle will also be quantified by measuring numbers of merozoites per mature schizont, merozoite invasion efficiency, use of alternative receptors for invasion, and cell cycle duration. The overall variance in each of the measured parameters and their associations with asexual multiplication rate will be tested.

3) How do the alternative major haplotypes of the gdv1 3'-intergenic region affect transcription and sexual commitment?
The levels of 3'-intergenic and antisense long non-coding RNA transcripts, in comparison with the sense transcripts of gdv1, will be analysed at basal levels and in response to gametocytogenesis induction in these isolates, and tested for differences between the two major gdv1 3'-intergenic allelic types. Complementary to this test association, allelic replacement genetic modifications will be performed to determine the regulatory effects of different parts of the intergenic sequence.

The benefit of this research will be primarily through the discovery of critical determinants of malaria parasite variation that are likely to affect transmission and virulence. This has relevance for designing and predicting the response to therapeutics and future vaccines, for long-term control and elimination.

Recent advances have highlighted how important it is to understand parasite adaptation for the design and implementation of effective interventions. This project will uncover important mechanisms of variation in parasites from different endemic populations. We will utilise novel methods to analyse the parasite development and gene expression determining sexual and asexual reproduction, which are the key phases of development in the blood leading to infection transmission and virulence respectively.

These results will be key to understanding malaria, and generating new ideas for effective vaccines and therapeutics. Results from this investigation will change our understanding of parasites as dynamic targets. This is essential for understanding mechanisms of parasite virulence, severe malaria, asymptomatic malaria, persistent chronic infection carriage, induction of sexual stage development and transmission.

The long-term impact on therapeutic and vaccine design should be significant, and will affect the ongoing mission of academic and disease control communities focusing on the following aspects of malaria:

- Epidemiology, control and parasite adaptation
- Clinical disease and transmission determinants
- New vaccine and drug candidate target identification
- Cellular and molecular mechanisms in parasites

This research represents a new component within the programmes of LSHTM and collaborating institutions that are well oriented towards impact on the global scientific and health community.