Biochemistry and Pharmacology - Theses
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ItemNo Preview AvailableUnderstanding virulence protein trafficking in the P. falciparum infected red blood cellCarmo, Olivia Maria Silva ( 2022)After invading the red blood cell (RBC), the malaria-causative parasite P. falciparum traffics an adhesin, erythrocyte membrane protein 1 (here referred to as EMP1), to the host cell surface. EMP1 is essential for parasite survival in vivo as it prevents splenic clearance. Moreover, variants of EMP1 that confer cytoadherence to cerebral or placental tissue can lead to fatal complications of the disease. In addition to EMP1, ~500 other parasite proteins are exported into the host cell compartment, localizing to parasite- induced membrane-bound and membraneless structures and knob-like protrusions at the RBC surface. These exported proteins do not resemble canonical trafficking machinery (e.g., ESCRT, SNARE, and Rab machineries), and there is no remnant secretory machinery for the parasite to co-opt, so how EMP1 is transported through the host cell cytoplasm remains unclear. Here we present functional characterization of two exported proteins with potential roles in EMP1 transport. First focusing on the gametocyte exported protein 7 (GEXP07), we found that GEXP07 localizes to the Maurer’s clefts, an intermediate compartment for EMP1 trafficking. In the absence of GEXP07, the clefts segment into smaller membrane- bound structures, the knobs are larger and clustered, and EMP1 transport to the host cell surface is reduced. The work confirms the critical role of Maurer’s clefts in EMP1 trafficking and reveals a previously unappreciated link between EMP1 transport and host cell remodeling. We also characterized the PfEMP1 trafficking protein 7 (PTP7). We found that PTP7 localizes to the Maurer’s cleft and associated structures, including vesicles and membraneless structures called J-dots. In the absence of PTP7, the clefts become decorated with budding vesicles - seemingly stalled in the process of fission. The knobs morphology is altered and forward trafficking of EMP1 from the cleft to the infected RBC surface is ablated. We show that the poly-asparagine repeat-containing C-terminal domain of PTP7 is essential for its function. The work leads to the intriguing suggestion that low complexity domains might be important for the function of these non-canonical trafficking proteins. We sought to further understand the physical processes that might drive trafficking of exported proteins and explored the role of intrinsically unstructured protein domains, which have been shown to drive some trafficking events in other organisms. As mobile, membrane-less structures, the J-dots resemble the phase separated biomolecular condensates observed in other eukaryotes. Here, we used an optogenetic technique in a heterologous mammalian cell system to explore the condensate-forming potential of full-length J-dot proteins and protein domains. We identified the central region of a protein called 0801 as having a propensity to phase separate. We generated sequence variants of this protein region and determined the molecular grammar responsible for condensate formation. This work points to the possibility that intrinsically unstructured protein domains could play a previously unrecognized role in protein trafficking and/or protein sequestration in P. falciparum. Overall, the work presented in this thesis adds new insights to our understanding of EMP1 trafficking. Also, in the context of the RBC cytoplasm which lacks canonical trafficking machinery, our preliminary findings regarding the phase separation capacity of some exported proteins may inform the wider field of protein transport. If validated in P. falciparum, our findings prompt a re-evaluation of the molecular requirements for coordinated protein transport in eukaryotes more broadly.