To invade its definitive host, the mosquito, the malaria parasite must cross the 
midgut peritrophic matrix that is composed of chitin cross-linked by 
chitin-binding proteins and then develop into an oocyst on the midgut basal 
lamina. Previous evidence indicates that Plasmodium ookinete-secreted chitinase 
is important in midgut invasion. The mechanistic role of other ookinete-secreted 
enzymes in midgut invasion has not been previously examined. De novo mass 
spectrometry sequencing of a protein obtained by benzamidine affinity column of 
Plasmodium gallinaceum ookinete axenic culture supernatant demonstrated the 
presence of an ookinete-secreted plasmepsin, an aspartic protease previously 
only known to be present in the digestive vacuole of asexual stage malaria 
parasites. This plasmepsin, the ortholog of Plasmodium falciparum plasmepsin 4, 
was designated PgPM4. PgPM4 and PgCHT2 (the P. gallinaceum ortholog of P. 
falciparum chitinase PfCHT1) are both localized on the ookinete apical surface, 
and both are present in micronemes. Aspartic protease inhibitors (peptidomimetic 
and natural product), calpain inhibitors, and anti-PgPM4 monoclonal antibodies 
significantly reduced parasite infectivity for mosquitoes. These results suggest 
that plasmepsin 4, previously known only to function in the digestive vacuole of 
asexual blood stage Plasmodium, plays a role in how the ookinete interacts with 
the mosquito midgut interactions as it becomes an oocyst. These data are the 
first to delineate a role for an aspartic protease in mediating Plasmodium 
invasion of the mosquito and demonstrate the potential for plasmepsin 4 as a 
malaria transmission-blocking vaccine target.