Periodic Reporting for period 2 - MALART (Cellular basis of Artemisinin resistance in malaria parasites)
Okres sprawozdawczy: 2023-06-01 do 2024-11-30
Reduced susceptibility of the human malaria parasite Plasmodium falciparum to the first line drug artemisinin and derivatives (ART) has spread in South East Asia and now is also spreading in Africa where the highest burden of malaria lies. While ART is typically given with a partner drug, resistance to partner drugs also exists, together jeopardising treatment and the general goal of reducing malaria-related deaths. Most cases with reduced susceptibility to ARTs contain mutations in a gene of the parasite encoding the protein Kelch13. In previous work we and others showed that Kelch13 is needed for the parasite to endocytose cytosol from the host red blood cell (consisting almost exclusively of haemoglobin). The parasite grows within human red blood cells and “feeds” on the host cell cytosol by an endocytic process (taking up host cell cytosol in membrane bound packets formed at the interface to the host cell) and digests the content in its food vacuole. This is critical for parasite survival, firstly, to obtain amino acids from digested haemoglobin and secondly, to make room for growth in the host cell. However, the iron containing part of haemoglobin can not be digested and is stored in the food vacuole of the parasite. This degradation product is known to activate ART, which only then becomes toxic for the parasite. In parasites where Kelch13 is less active, such as in the mutated forms found in resistant parasites, less ART is activated, reducing the susceptibility of the parasite to this drug. However, it will also reduce the amino acids taken in, hence imposing a fitness cost on the parasite.
All of this highlighted that we
1. do not understand the endocytosis process in the parasite: how are host cell cytosol containers formed, which proteins are involved (K13 one of the first molecular players), how is endocytosis regulated, does it respond to nutrient cues, is it connected to developmental switches and speed of growth? (part 1)
2. do not know how Kelch13 influences endocytosis as this protein is not a typical vesicle trafficking protein (part 2)
3. do not know whether the fitness cost limits the level of resistance the parasite can achieve and does this impose constraints and compensatory responses in the parasite: ART resistance is only a partial resistance and it is unclear if parasites could become more resistant. Finally, the impact of the fitness cost on the parasite and potential constraints this imposes on the ART resistant parasites and how this potentially could be exploited is unclear (part 3)
In this project we are addressing these questions by unravelling the fundamental, so far poorly understood vesicle trafficking pathway that is critical for ART resistance, thereby bridging basic cell biology and a topic of high clinical relevance.
All of this highlighted that we
1. do not understand the endocytosis process in the parasite: how are host cell cytosol containers formed, which proteins are involved (K13 one of the first molecular players), how is endocytosis regulated, does it respond to nutrient cues, is it connected to developmental switches and speed of growth? (part 1)
2. do not know how Kelch13 influences endocytosis as this protein is not a typical vesicle trafficking protein (part 2)
3. do not know whether the fitness cost limits the level of resistance the parasite can achieve and does this impose constraints and compensatory responses in the parasite: ART resistance is only a partial resistance and it is unclear if parasites could become more resistant. Finally, the impact of the fitness cost on the parasite and potential constraints this imposes on the ART resistant parasites and how this potentially could be exploited is unclear (part 3)
In this project we are addressing these questions by unravelling the fundamental, so far poorly understood vesicle trafficking pathway that is critical for ART resistance, thereby bridging basic cell biology and a topic of high clinical relevance.
For part 1 we so far obtained a comprehensive set of the proteins located at the structure where host cell haemoglobin is internalised into the parasite, the so called cytostome. We showed for several of these proteins that they are needed for this process and that they reduce the susceptibility of the parasites to ART when their activity is reduced (fitting with the model that reduced uptake reduces also ART resistance). Many of these proteins are very different from endocytosis proteins in other organisms such as humans, hence they are potential targets for drugs as such drugs would likely be specific for the parasite and not harm the human host. Further this work also showed that a particular actin motor is needed for endocytosis, bringing actin into the model for this process. Finally, we could clarify the route of uptake, showing that function at the cytostome leads to host cell cytosol packets that are transported to the parasite’s food vacuole, connecting the two so far known pieces in the pathway. Together this indicates that the initial phase of uptake, when haemoglobin filled vesicles are formed, is highly parasite-specific and atypical (compared to model organisms) whereas the transport of these containers to the food vacuole contains elements conserved in other organisms such as humans.
Work for part 2 has not yet made a decisive advance, but we have a general grasp which of the main parts of Kelch13 are needed for its function and that its abundance in the cell and location at the cytostome are critical for the rate of endocytosis and ART resistance.
For part 3 we found that the fitness cost of resistance likely prevents the parasite from reaching higher levels of resistance. While we were able to increase resistance by constant ART exposure cycles, resistance plateaued and the parasites had an even further slowed development cycle, indicating a higher fitness cost. We also found that parasites with two known resistance mutations in the gene encoding Kelch13 do not become more resistant, suggesting that combination of resistance mutations (at least for this combination) might not be a threat to result in hyperresistant parasites. Moreover, we found that other cytostome proteins, while providing resistance when mutated or partially inactivated, result in a very high fitness cost. This is likely due to the fact that Kelch13 is unique in influencing endocytosis only at a specific life cycle stage while many of the other endocytosis proteins function also in other parts of the development of the parasite in the red blood cell. The proportional increase in fitness cost with resistance fuels the hope that the partial resistance seen in endemic settings might not easily increase further.
Work for part 2 has not yet made a decisive advance, but we have a general grasp which of the main parts of Kelch13 are needed for its function and that its abundance in the cell and location at the cytostome are critical for the rate of endocytosis and ART resistance.
For part 3 we found that the fitness cost of resistance likely prevents the parasite from reaching higher levels of resistance. While we were able to increase resistance by constant ART exposure cycles, resistance plateaued and the parasites had an even further slowed development cycle, indicating a higher fitness cost. We also found that parasites with two known resistance mutations in the gene encoding Kelch13 do not become more resistant, suggesting that combination of resistance mutations (at least for this combination) might not be a threat to result in hyperresistant parasites. Moreover, we found that other cytostome proteins, while providing resistance when mutated or partially inactivated, result in a very high fitness cost. This is likely due to the fact that Kelch13 is unique in influencing endocytosis only at a specific life cycle stage while many of the other endocytosis proteins function also in other parts of the development of the parasite in the red blood cell. The proportional increase in fitness cost with resistance fuels the hope that the partial resistance seen in endemic settings might not easily increase further.
The finding that resistance seems to be consistently limited by the fitness cost indicates that the Kelch13-dependent mechanism can not lead to a sterile resistance due the reduction of nutrients that comes with it. This is critical to know and might also help finding partner drugs that have a synergistic effect in resistant parasites such as drugs acting on haemoglobin digestion in the parasite’s food vacuole. We are currently testing what could be the cause of the increased, but plateaued, resistance in our serially selected parasites and also how the parasite tries to compensate for the lowered nutrient to further understand the constraints the parasite is under and the ways it may escape to further resistance.
For the first and second part we are further defining the pathway of endocytosis, the proteins involved, how the function mechanistically and how endocytosis levels are regulated. We have initial evidence for a connection of the parasite development program and endocytosis which would be critical to understand the basic biology of the parasite but also potentially for ART resistance.
For the first and second part we are further defining the pathway of endocytosis, the proteins involved, how the function mechanistically and how endocytosis levels are regulated. We have initial evidence for a connection of the parasite development program and endocytosis which would be critical to understand the basic biology of the parasite but also potentially for ART resistance.