Leishmania subtilisin is a maturase for the trypanothione reductase system and contributes to disease pathology

Abstract

Proteases are a ubiquitous group of enzymes that play key roles in the life cycle of parasites, in the host-parasite relationship, and in the pathogenesis of parasitic diseases. Furthermore, proteases are druggable targets for the development of new anti-parasitic therapy. The subtilisin protease (SUB; Clan SB, family S8) of Leishmania donovani was cloned and found to possess a unique catalytic triad. This gene was then deleted by gene knock-out, which resulted in reduced ability by the parasite to undergo promastigote to amastigote differentiation in vitro. Electron microscopy of SUB knock-out amastigotes revealed abnormal membrane structures, retained flagella, and increased binucleation. SUB-deficient Leishmania displayed reduced virulence in both hamster and murine infection models. Histology of spleens from SUB knock-out-infected hamsters revealed the absence of psammoma body calcifications indicative of the granulomatous lesions that occur during Leishmania infection. To delineate the specific role of SUB in parasite physiology, two-dimensional gel electrophoresis was carried out on SUB(-/-) versus wild-type parasites. SUB knock-out parasites showed altered regulation of the terminal peroxidases of the trypanothione reductase system. Leishmania and other trypanosomatids lack glutathione reductase, and therefore rely on the novel trypanothione reductase system to detoxify reactive oxygen intermediates and to maintain redox homeostasis. The predominant tryparedoxin peroxidases were decreased in SUB(-/-) parasites, and higher molecular weight isoforms were present, indicating altered processing. In addition, knock-out parasites showed increased sensitivity to hydroperoxide. These data suggest that subtilisin is the maturase for tryparedoxin peroxidases and is necessary for full virulence.

FIGURE 1.

The catalytic cores of the Leishmania subtilisins were compared with the cores of other Clan SB, family S8 family members, as determined by Pfam. Core sequences were aligned using ClustalW2 (EMBL-EBI). The Leishmania SUBs group with the subfamily S8A proteases, which include the eukaryotic Site- 1 peptidases and the bacterial subtilisins. This distinguishes Leishmania SUB from the Toxoplasma and Plasmodium SUBs and from the subfamily S8B kexins and furins.

FIGURE 2.

Successful deletion of both genomic copies of the SUB gene was determined by Southern blot analysis. Digested genomic DNA from wild-type, single-copy, and double-copy knockouts were analyzed at the SUB locus for the presence of either the wild-type locus or the knock-out cassette. The blot was stripped and re-probed for the core of the SUB gene itself to ensure that the gene was not relocated to another site in the genome.

FIGURE 3.

Wild-type, SUB^+/−, and −/− L. donovani promastigotes were cultured successfully at 27 °C in M199. To test for the ability to differentiate into axenic amastigotes, stationary phase (day 5 post-split) promastigotes of each culture were split 1:10 in FBS at 37 °C. Wild-type and SUB^+/− parasites differentiated readily, however SUB^−/− did not. The SUB^−/− parasites remained as elongated, flagellated spindles. These cells did not form aggregates of cells typically seen in axenic amastigote cultures.

FIGURE 4.

Wild-type axenic amastigotes (A) had normal rounded cell bodies measuring ∼3 μm in diameter (scale bar = 1 μm) with no external flagellum and a condensed electron-dense kinetoplast. SUB^−/− axenic amastigotes (B–D) exhibited many abnormalities. Although most of these cells had rounded cell bodies, many were still elongated and spindle-shaped (D). Those that were rounded often had invaginations in the plasma membrane (B). Additionally, many of these cells were binucleated (C). Unlike the wild-type amastigotes, the SUB^−/− cells also had multiple flagellar cross-sections, including flagella appearing in the cytoplasm (B), outside of their expected location within the flagellar pocket (N = nucleus, K = kinetoplast, FP = flagellar pocket, and F = flagellum).

FIGURE 5.

Wild-type L. donovani had high levels of TXNPx1 and -3 forming a single doublet of spots (WT spots 4 and 5, left). The level of TXNPx2 was low (spot 3) and Prx was not detectable (spot 1). In the SUB^−/− parasites (right), the wild-type TXNPx1/3 doublet decreased (SUB^−/− spots 4 and 5) and two new TXNPx1/3 doublets were present at a higher molecular weight (spots 6 and 7) and at a higher molecular weight with a lower pI (spots 8 and 9). Prx levels (spots 1 and 2) were elevated in these parasites.

FIGURE 6.

Leishmania major promastigote replication was measured in the presence of varying concentrations of tert-butylhydroperoxide. Wild-type (black squares) and SUB-deficient (dark gray squares) were grown to stationary phase and counted on a Multisizer 3 Coulter Counter. Values are expressed as the percent culture density relative to the untreated controls.

FIGURE 7.

BALB/c mice (n = 5) were infected subcutaneously in the left hind footpads with wild-type (solid circle) or SUB^+/− (empty circle) parasites. Footpad swelling was measured weekly and the footpad thickness was plotted over time. Footpad size was significantly larger (p = 0.001) in WT infections after 40 days PI. Error bars indicate the ± S.D. between the 5 mice in each group. The mice were sacrificed at 180 days post infection. No swelling was observed in the right hind (uninfected) footpads. Male Golden Syrian hamsters (n = 3) were infected intraperitoneally with wild-type or SUB^−/− L. donovani. At 200 days post-infection, the hamsters were sacrificed, and their spleens were sectioned, H&E-stained, and histologically examined. All wild-type-infected hamsters' spleens (B and C) contained psammoma body calcifications (arrows). No psammoma bodies were observed in the spleens of hamsters infected with SUB-deficient Leishmania (D and E).