Biology
Dr. Budhaditya Mukherjee
Department of Microbiology and Molecular Medicine, Faculty of Medicine - University of Geneva, Switzerland
Abstract:
The phylum Apicomplexa consists of largest group of intracellular protozoan pathogens causing fatal disease to human and economically important animals. Central to the survival and dissemination of these obligate intracellular parasites is their capacity to actively invade and egress from host cells. An arsenal of secretory proteins is sequentially discharged from specialized secretory organelles during egress (micronemes) and invasion (micronemes & rhoptries) that act as perforins, adhesins, proteases, or kinases and allows these parasites to complete their life cycle. Host attachment triggers the discharge of rhoptry proteins, (RONs & ROPs) that critically participate in the entry by forming the moving junction (MJ) and subverting of host cellular functions, respectively.
We recently showed that the Toxoplasma gondii aspartyl protease 3 (TgASP3) serves as essential maturase for micronemes and rhoptry proteins and thus plays a crucial role in invasion and egress (Elife, 2017). Remarkably, orthologs of TgASP3 in the malaria parasites, Plasmepsin IX (PMIX) and Plasmepsin X (PMX), fulfil this fundamental functions in all the invasive stages of malaria parasites (Science, 2018).
Potent hydroxyl-ethylamine scaffold-based peptidomimetic inhibitor 49c, targets this particular class of aspartyl proteases (TgASP3/PMIX/PMX) involved in invasion and egress without affecting similar other apicomplexan aspartyl proteases involved in hemoglobin degradation or protein export. The flap/flap-like structures of aspartyl protease are highly flexible and critically modulate the binding cavity access. Molecular docking of 49c into active site of TgASP3/PMIX/PMX predicted that a conserved residue (F344) could account for the sensitivity of this class of apicomplexan aspartyl proteases towards 49c. F344Y mutation in TgASP3 resulted in ten-fold increase in IC50 towards 49c, without affecting the sensitivity towards closely related compound 49b. Selection of mutagenized T. gondii mutants resistant to 49c, reproducibly converted F334 to a cysteine residue (F344C). F344C mutants exhibit similar IC50 as F344Y mutation without revealing any obvious steric clashes/hindrance with 49c suggesting a more complex mechanism of resistance. It is not clear why F344C mutation is favoured without conferring significant increase in resistance; it is plausible that F344C mutation allows for better substrates accessibility as compare to F344Y mutation. Failure to generate 49c-resistant mutants in Plasmodium in vivo further supports this notion of dual targeting of 49c in Plasmodium parasites. Concordantly, r-PMIX/PMX with critical mutation in phenylalanine residue resulted in 2-15 fold increase in selectivity towards 49c, confirming a conserved selection-mode for malaria proteases (EMBO J, 2018).
Comparison of proteome of wild type and aspartyl protease-depleted parasites have identified large number of novel substrates specific for these aspartyl proteases and presently we are trying to characterize them to explain egress and invasion defects in apicomplexan parasites.
