Apicomplexan genomes encode multiple pepsin-family aspartyl proteases (APs) that phylogenetically cluster to six independent clades (A to F). Such diversification has been powered by the function-driven evolution of the ancestral apicomplexan AP gene and is associated with the adaptation of various apicomplexan species to different strategies of host infection and transmission through various invertebrate vectors. To estimate the potential roles of Babesia APs, we performed qRT-PCR-based expressional profiling of Babesia microti APs (BmASP2, 3, 5, 6), which revealed the dynamically changing mRNA levels and indicated the specific roles of individual BmASP isoenzymes throughout the life cycle of this parasite. To expand on the current knowledge on piroplasmid APs, we searched the EuPathDB and NCBI GenBank databases to identify and phylogenetically analyse the complete sets of APs encoded by the genomes of selected Babesia and Theileria species. Our results clearly determine the potential roles of identified APs by their phylogenetic relation to their homologues of known function-Plasmodium falciparum plasmepsins (PfPM I-X) and Toxoplasma gondii aspartyl proteases (TgASP1-7). Due to the analogies with plasmodial plasmepsins, piroplasmid APs represent valuable enzymatic targets that are druggable by small molecule inhibitors-candidate molecules for the yet-missing specific therapy for babesiosis.

• Publication type
• Journal Article MeSH

Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite's ability to modulate host signalling pathways and immune responses.

• MeSH
• Aspartic Acid Proteases metabolism   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Fluorescent Antibody Technique MeSH
• Gene Knockout Techniques MeSH
• Golgi Apparatus enzymology   MeSH
• Host-Parasite Interactions physiology   MeSH
• Cells, Cultured MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Toxoplasma enzymology   pathogenicity   MeSH
• Toxoplasmosis enzymology   MeSH
• Transfection MeSH
• Microscopy, Electron, Transmission MeSH
• Protein Transport MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Therapeutic agents with novel mechanisms of action are urgently needed to counter the emergence of drug-resistant infections. Several decades of research into proteases of disease agents have revealed enzymes well suited for target-based drug development. Among them are the three recently validated proteolytic targets: proteasomes of the malarial parasite Plasmodium falciparum, aspartyl proteases of P. falciparum (plasmepsins) and the Sars-CoV-2 viral proteases. Despite some unfulfilled expectations over previous decades, the three reviewed targets clearly demonstrate that selective protease inhibitors provide effective therapeutic solutions for the two most impacting infectious diseases nowadays-malaria and COVID-19.

• MeSH
• Aspartic Acid Endopeptidases metabolism   MeSH
• COVID-19 enzymology   metabolism   MeSH
• COVID-19 Drug Treatment MeSH
• Protease Inhibitors pharmacology   MeSH
• Humans MeSH
• Malaria drug therapy   enzymology   metabolism   MeSH
• Plasmodium falciparum drug effects   pathogenicity   MeSH
• Proteasome Endopeptidase Complex drug effects   MeSH
• SARS-CoV-2 drug effects   pathogenicity   MeSH
• Drug Development methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Over the last decade, research on the most studied parasite, Plasmodium falciparum, has disclosed significant findings in protease research. Detailed descriptions of the individual roles of protease isoenzymes from various protease classes encoded by the parasite genome have been elucidated, along with their functional and biochemical characterizations. These insights have enabled the development of innovative chemotherapy using low molecular weight inhibitors targeting specific molecular sites. Progress has been made in understanding the proteolytic cascade associated with the apical complex, particularly the roles of aspartyl proteases plasmepsins IX and X as master regulators. Additionally, advancements in direct and alternative methods of proteasome inhibition and expression regulation have been achieved. Research on digestive/food vacuole-associated proteases, with a focus on essential metalloproteases, has also seen significant developments. The rise of extensive genomic datasets and functional genomic tools for other parasitic organisms now allows these approaches to be applied to the study and treatment of other, less known parasitic diseases, aiming to uncover specific biological mechanisms and develop innovative, less toxic chemotherapies.

• MeSH
• Antimalarials * pharmacology   therapeutic use   MeSH
• Protease Inhibitors * therapeutic use   MeSH
• Humans MeSH
• Malaria drug therapy   MeSH
• Plasmodium falciparum * enzymology   drug effects   genetics   MeSH
• Peptide Hydrolases metabolism   genetics   MeSH
• Malaria, Falciparum drug therapy   parasitology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH