Project Portugal 2030

Summary

The goal of this project lies in deciphering molecules and mechanisms that allow Plasmodium sporozoites (SPZ), to specifically halt their circulatory dissemination at the liver, a critical step for subsequent hepatocyte infection. Before crossing the sinusoidal barrier, two events might be at stake: initial recognition of the liver vasculature and a subsequent productive interaction, which based on our unpublished data, might require gliding motility for complete retention of SPZ within the sinusoids (Fig1-3). To achieve this, we propose a multifaceted approach, combining live-image-based assays with cutting-edge molecular replacement technology in parasites for functional studies, with three main aims in mind. In aim 1, we will investigate the contribution of SPZ gliding motility and the interplay with the host for the homing to liver using rodent malaria models and will expand the study to the human infecting P. falciparum SPZ. With rodent malaria parasites, we will conduct intravital microscopy studies in the flk1-GFP transgenic mice, which express GFP in endothelial cells and allow perfect delineation of the lumen of the sinusoid, to evaluate how SPZ with impaired gliding motility, either genetically or pharmacologically, interact with the liver sinusoids [11]. We will demonstrate the contribution of TRAP and SPZ gliding motility in this process. Additionally, we will explore the species specificity of SPZ homing to the liver. We hypothesize that the homing and crossing of the liver sinusoidal barrier is, as the exit of SPZ from the skin [22], a species-specific independent process. In aim 2, we seek to broaden our understanding of molecules and mechanisms involved in SPZ homing by conducting a comprehensive functional screening of barcoded gene knockout SPZ mutants, employing barcode counting on a next-generation sequencer (barseq). Leveraging the PlasmoGEM resource [23], we will prioritize knockouts of approximately 100 genes expressed in SPZ [24], previously implicated in infection outcomes [25]. This innovative screening approach involves quantifying mutant SPZ that, following blood dissemination in mice, fail to home to the liver and remain in circulation. The genes for which the deletion resulted in the most severe homing defective phenotypes will be analysed using bioinformatic tools and organized in pathways or biological processes. Subsequently, the most infection-defective mutants will undergo detailed investigations using a combination of in vitro studies and live imaging. Using this novel functional approach, we will identify and characterize novel targets in SPZ. In aim 3, we will explore the role of CSP in SPZ homing to liver. Given CSP's essential role in SPZ formation, we will assess whether interfering with CSP binding capacity affects homing, by employing infections in mice using CSP-specific monovalent fragment antigen-binding (Fab). These were generated using Fab-phage display technology and are already available in my lab (Fig4). Furthermore, we will investigate the impact of post-translational proteolytic cleavage of CSP [19], on SPZ homing efficiency, utilizing SPZ lacking specific regions of CSP in homing assays. The outcomes of this interdisciplinary project will significantly advance our mechanistic understanding of a critical step in malaria SPZ infectivity, thereby laying the groundwork for the development of future interventions aimed at preventing infection.