UCSF

Proteases are a ubiquitous group of enzymes that play key roles in the life cycle of parasites and in the host-parasite relationship. They are important targets for the development of new anti-parasitic therapy. Protozoan parasites like Leishmania predominantly express Clan CA cysteine proteases. It was therefore unexpected to find a high level of serine protease activity in Leishmania donovani. Oligopeptidase B (OPB) was identified as the responsible enzyme. This was confirmed by OPB gene knockout, which resulted in the disappearance of the major serine protease activity. To delineate the role of OPB in parasite physiology, two-dimensional gel electrophoresis was carried out on OPB -/- versus wildtype parasites. Four protein species were significantly elevated in OPB -/- parasites, and all four were identified as enolase. Aside from its classic role in carbohydrate metabolism, enolase is known to bind plasminogen and function as a virulence factor for several infectious microbes. As expected, there was a striking alteration in macrophage responses to Leishmania when OPB was deleted. While wildtype parasites elicited little response from infected macrophages, OPB -/- parasites induced a massive upregulation in transcription. OPB -/- parasites displayed decreased virulence in the murine infection model. Additionally we have investigated the subtilisin (SUB) of Leishmania because it is a single-copy gene and subtilisins play important roles in virulence of other protozoa. Leishmania subtilisin was deleted by gene knockout, which resulted in reduced ability to undergo promastigote-amastigote differentiation in vitro. EM of SUB knockout amastigotes revealed abnormal membrane structures, retained flagella, and binucleation. SUB deficient Leishmania displayed reduced virulence in hamster and murine infection models. Histology of spleens from SUB knockout-infected hamsters revealed the absence of psammoma body calcifications. To delineate the role of SUB in parasite physiology, two-dimensional gel electrophoresis was carried out on SUB -/- versus wildtype parasites. SUB knockout parasites showed altered regulation of the trypanothione reductase system. Trypanosomatids lack glutathione reductase, and rely on the novel trypanothione reductase system to detoxify ROSs and maintain redox homeostasis. The predominant tryparedoxin peroxidase was decreased in SUB -/- parasites, and higher molecular weight isoforms of the enzyme were present, suggesting altered processing.