mTRAP: Gene knockouts trap malaria in the erythrocyte

Malarial TRAP family proteins were thought to function during cell invasion, with the merozoite form MTRAP involved in asexual erythrocytic infection. However, a recent study of gene disruption using knockouts and the CRISPR-Cas9 system reveal unexpected roles for the MTRAP protein in cell egress and gamete development. 

The TRAP (thrombospondin-related anonymous protein) family are conserved in various apicomplexan parasites causing severe human disease, including malaria (Plasmodium spp), and toxoplasmosis (Toxoplasma spp). Previous research studying the TRAP protein family had suggested functions during cell invasion and gliding motility ². Whilst this may hold true for some of the protein family members, research undertaken by Bargieri et al ¹ has revealed the merozoite TRAP protein (MTRAP) operates in an unexpected way.

Using gene knockouts in the malaria animal model Plasmodium berghei, and the CRISPR-Cas9 system in human Plasmodium falciparum, the research suggests that MTRAP deficiency results in a transmission block. Without MTRAP, neither male nor female gametocytes are able to egress from erythrocytes in the mosquito, which prevents zygote formation and further transmission. It had been thought MTRAP was expressed in the Plasmodium asexual merozoite, responsible for the erythrocytic cycle, where it mediated adhesion and invasion of the red blood cell. This theory was primarily based on previous research suggesting MTRAP localised in the merozoite apical microneme organelles, interacting with the parasite invasion machinery. However this work had been unable to present evidence of MTRAP binding directly to erythrocytes^{2, 5}, which is now explained by the findings of Bargieri et al.

In P. berghei, the authors first noticed inability to express MTRAP showed no effect on asexual blood stage parasite growth. When investigating this finding in both wildtype and knockout Plasmodium spp, immunofluorescence assays using polyclonal antibodies identified MTRAP expression at higher levels in the sexual gametocyte stages. Using fluorescent protein expression in gametocytes revealed that MTRAP deficiency correlated with non-formation of ookinetes or oocysts in the mosquito midgut – and unexpectedly that MTRAP has a role in the sexual stages of the malaria lifecycle.

Testing the applications of this research involved moving from an animal model to the human species malaria Plasmodium falciparum. Using the CRISPR-Cas9 system to disrupt the MTRAP locus showed no change in blood stage parasitaemia, with MTRAP expression instead being strongly detected in gametes unable to rupture the parasitophorous vacuole membrane. 

Figure 1: Malaria lifecycle, with inset results from MTRAP research in Plasmodium spp ^{1, 4.}
The research by Bargieri et al suggests MTRAP functions in the sexual stages of malaria, with protein removal preventing parasite transmission or fertilisation. (A) In the blood stages, identical parasite growth curves were found for both the wildtype MTRAP and the gene knockouts, suggesting the protein does not play a significant role. (p.i = post infection). (B) The research found also found normal levels of circulating gametocytes in the mouse bloodstream. (C) P. falciparum gametocyte staining with DAPI for DNA  (blue) and Band3 for the erythrocytic membrane (green) shows activated gametocytes remaining inside the erythrocytic membrane (D) Gene knockouts of MTRAP in Plasmodium berghei were found to negatively influence both ookinete and oocyst formation in the mosquito host.

In wild type parasites, exflagellation of the male macrogamete occurs inside the erythrocytic parasitophorous vacuole. Before egress from the erythrocyte, the vacuole is ruptured and male gametes form exflagellation centres on the inner red blood cell membrane. Activated MTRAP^KO  gametes were found to form motile flagella but did not form exflagellation centres in either P. berghei or P. falciparum. Microscopy of gamete activation found the knockouts remained inside the parasitophorous vacuole, and the authors believe this stems from an inability to disrupt the vacuole membrane.  Further evidence was given to this theory by complementing defective mutants with an episomally expressed MTRAP, which resulted in partial restoration of the parasite sexual cycle.

Emerging resistance to commonly used antimalarial compounds and insecticides pose a risk to malaria control efforts, and there is not yet an efficacious commercially available vaccine ^{4, 5}.  Although this research does not suggest MTRAP as a candidate for vaccines blocking erythrocytic invasion, the protein could be explored to block transmission. Further research into MTRAP’s role in parasitophorous vacuole disruption and gamete egress is needed to understand its potential role in a vaccine. It may be that MTRAP does indeed have roles in parasite actin motors and motility, although in a different stage to the parasite lifecycle than previously expected. Whilst further work still needs to be performed exploring MTRAP function, this research clearly demonstrates the remarkable utility of the CRISPR-Cas9 system in malaria.

References

1. Bargieri, D.Y. et al. Plasmodium Merozoite TRAP Family Protein Is Essential For Vacuole Membrane Disruption and Gamete Egress From Erythrocytes. Cell Host & Microbe 20, 619-630 (2016).
2. Baum, J. et al. A conserved molecula motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites. Journal of Biological Chemistry 281, 5197-5208 (2006).
3. Kehrer, J., Frischknecht, F. and Mair, G. R. Proteomic analysis of the Plasmodium berghei gametocyte egressosome and vesicular bilD of osmiophilic body proteins identifies merozoite TRAP-like protein (MTRAP) as an essential factor for parasite transmission. Molecular and Cellular Proteomics 15, 2852-2862. (2016).
4. Outtara, A and Laurens, M.B. Vaccines against malaria. Clinical Infectious Diseases 60, 930-936. (2015).
5. Cowman, A.F., Healer, J.m Marapana, D. and Marsh, K. Malaria: Biology and Disease. Cell 167, 610-624. (2016).