Periodic Reporting for period 1 - NotToKill-NotToDie (Unrevealing dry season Plasmodium falciparum replication biology)
Reporting period: 2019-07-01 to 2021-06-30
The Action with acronym “NotToKill-NotToDie” looks into how P. falciparum parasites are able to survive during the long 6 moths’ dry season in of certain malaria endemic regions without being cleared by the immune system. P. falciparum persists at very low parasitemia in close to one third of the children during the dry season without causing symptoms, and representing an essential reservoir of parasites needed to restart transmission as the rainy season ensues and mosquitos re-appear. The research questions on the mechanism/s underneath this important phenomenon, with a special emphasis on unrevealing a potential alteration in the parasite’s intrinsic capacity to replicate. Such topic is important because society needs to propose new and innovative routes to tackle malaria; and prophylactic drugs during the dry season when parasite numbers are low, and the prevalence of infected people much lower than in the transmission season, seems a good way forward for alternative treatment strategies. Not only is there widespread lack of knowledge on the parasite’s intrinsic biology leading to asymptomatic infections under this scenario, but also an uncertainty on how under similar exposure to malaria some people carry parasites triggering severe malaria outcomes during the transmission season while others just remain asymptomatic. Although working during the dry season in a malaria endemic country poses a challenge i, we have been able to address the topic of parasite replication through a variety of innovative approaches. Objectives of this MSCA have been to (1) Characterize dry-season parasites through expression kinetics of parasite replication-associated genes and their phenotype, and (2) Investigate PfCRK4 regulation of P. falciparum cell cycle progression, and its role in dry season transcription modulation. A parallel goal of the MSCA Individual Fellowship (Reintegration) is to foster the development of the individual researcher, achieve a productive international collaboration and disseminate the work in the local society.
WP1 comprised visits to ICER-Mali for the collection of samples whose use yielded 4 conference presentations and 2 publications. Project was managed through WP2. In WP3 he exceeded goals, not only by unrevealing that replication-related transcriptional RNA-seq signatures were due to increased time in circulation of the infected red blood cells before cytoadhering to the microvasculature ; and leading to first author publication. But also, by expanding these findings to shed light into previously published misleading transcriptomes analysis regarding different clinical presentations of malaria, and that has recently led to another publication in which the fellow is first author, and another one underway. In WP4, the fellow attempted to obtain P. falciparum mutants to perform gene expression-phenotype associations through gene deletion of 10 PfCRK4-modulated genes involved in parasite replication, which were also found to be dysregulated in parasites during the dry season. All genes were found to be essential as no knock-out was achieved. The finding that the peak of expression of all the replication-related genes tested was in the middle of the 48h life cycle, suggest that the dysregulation of replication-related genes in the dry season is indeed cell cycle dependent. In WP5/WP6 he attended 8 multi-day conferences where he presentet results of his work ; 4 training courses and workshops; biweekly DZIF malaria-related seminars; and weekly department seminars. He provided leadership in presearch, presented orally and through posters in international symposiums and conducted 8 seminars for researchers and educators; he provided supervision and mentoring for students and disseminate his expertise to society twice through the EU “Researchers Night”.
Overview: With no mosquitoes around during the dry season, P. falciparum must live for up to six months inside people. This offers the human immune system an opportunity to eliminate this parasitic. To grow inside a person, the parasite pops out a protein that attaches the infected cell to the walls of blood vessels. This allows the parasite to multiply and resist going through the spleen. Parasite drops to low numbers during the dry season which might also be a strategy used by the parasite to keep a low profile. The parasite pops out different grabbers during the dry season, that do not stick so well to the walls of blood vessels, allowing more parasite to be filtered out by the spleen. The immune system seems not to see really low numbers of the parasite. P. falciparum parasites includes about 60 genes for the main grabber protein (60 versions of the protein). This makes it harder for the body to recognise and build immunity to its foe. The parasite a pulls out its stickiest protein first, but once the immune system responds and blocks that, it shuffles through dozens of less stickygrabbers. Once the rains come, the next person will get a form of the parasite that again adheres strongly and multiplies rapidly inside. For this reason, drugs are given to younger children during the rain season as they are the most vulnerable to malaria. But we wonder if it might be useful to give prophylactic drugs during the dry season, when parasite numbers are low and lower prevalence of infected people. This could be an opportunity to kill off the parasite during the dry season. This strategy is as yet unproven.
Overview: With no mosquitoes around during the dry season, P. falciparum must live for up to six months inside people. This offers the human immune system an opportunity to eliminate this parasitic. To grow inside a person, the parasite pops out a protein that attaches the infected cell to the walls of blood vessels. This allows the parasite to multiply and resist going through the spleen. Parasite drops to low numbers during the dry season which might also be a strategy used by the parasite to keep a low profile. The parasite pops out different grabbers during the dry season, that do not stick so well to the walls of blood vessels, allowing more parasite to be filtered out by the spleen. The immune system seems not to see really low numbers of the parasite. P. falciparum parasites includes about 60 genes for the main grabber protein (60 versions of the protein). This makes it harder for the body to recognise and build immunity to its foe. The parasite a pulls out its stickiest protein first, but once the immune system responds and blocks that, it shuffles through dozens of less stickygrabbers. Once the rains come, the next person will get a form of the parasite that again adheres strongly and multiplies rapidly inside. For this reason, drugs are given to younger children during the rain season as they are the most vulnerable to malaria. But we wonder if it might be useful to give prophylactic drugs during the dry season, when parasite numbers are low and lower prevalence of infected people. This could be an opportunity to kill off the parasite during the dry season. This strategy is as yet unproven.
This MSCA has pushed the frontiers of the understanding of parasites in the context of a dry season malaria transmission setting and how parasitemia is controlled in chronic infections. Moreover, the fellow has proposed a paradigm shift on the mechanisms of pathogenesis in malaria by revealing through transcriptional signatures that parasite circulation time associates with growth potential and parasite virulence, influencing disease outcome, and potentially highlight cytoadhesion dynamics as a major force driving the clinical prognosis of malaria. Following infection, individuals can remain asymptomatic, present with mild fever in uncomplicated malaria cases, or show one or more severe malaria symptoms. Several studies have investigated associations between parasite transcription and clinical severity, but no broad conclusions have yet been drawn. This MSCA allowed the Fellow to develop agility with many different research methodologies and promote best practices to the larger community. The fellow has applied apply a series of bioinformatic approaches based on P. falciparum’s tightly regulated transcriptional pattern during its ~48-hour intraerythrocytic developmental cycle (IDC) to publicly available transcriptomes of parasites obtained from malaria cases of differing clinical severity across multiple studies. Through the Fellow’s research it has been shown that within each IDC, the circulation time of infected erythrocytes without sequestering to endothelial cells decreases with increasing parasitaemia or disease severity. Accordingly, we found that the size of circulating infected erythrocytes is inversely related to parasite density and disease severity. We proposed that enhanced adhesiveness of infected erythrocytes leads to a rapid increase in parasite burden, promoting higher parasitaemia and increased disease severity.