The oligopeptidase B of Leishmania regulates parasite enolase and immune evasion

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 targets for the development of new anti-parasitic therapy. Protozoan parasites like Leishmania predominantly express Clan CA cysteine proteases for key life cycle functions. It was therefore unexpected to find a high level of serine protease activity expressed by Leishmania donovani. Purification of this activity followed by mass spectrometry identified oligopeptidase B (OPB; Clan SC, family S9A) as the responsible enzyme. This was confirmed by gene knock-out of OPB, which resulted in the disappearance of the detected serine protease activity of Leishmania extracts. To delineate the specific role of OPB in parasite physiology, proteomic analysis was carried out on OPB(-/-) versus wild type parasites. Four protein species were significantly elevated in OPB(-/-) parasites, and all four were identified by mass spectrometry as enolase. This increased enolase was enzymatically inactive and associated with the parasite membrane. Aside from its classic role in carbohydrate metabolism, enolase was recently found to localize to membranes, where it binds host plasminogen and functions as a virulence factor for several pathogens. As expected, there was a striking alteration in macrophage responses to Leishmania when OPB was deleted. Whereas wild type parasites elicited little, if any, response from infected macrophages, OPB(-/-) parasites induced a massive up-regulation in gene transcription. Additionally, these OPB(-/-) parasites displayed decreased virulence in the murine footpad infection model.

FIGURE 1.

Identification of high serine protease activity in Leishmania lysate. A, the soluble fraction of L. donovani lysate was tested for protease activity using a Z-PR-AMC substrate. Activity assays were performed at pH 5.5 (white bar) and at 8.0 (black bar), pH levels that are generally preferred by cysteine and serine proteases, respectively. The activity observed was tested for sensitivity to the cysteine protease inhibitor E-64 (10 μm) and the serine protease inhibitor PEFABLOC (2 mm). The DMSO concentration was 1%. Activities were normalized to the protease activity observed at the indicated pH without inhibitor. B, OPB activity was compared between three species of Leishmania and T. cruzi. Insect vector stages of the parasites (Leishmania promastigotes and T. cruzi epimastigotes, white bar) were compared with the vertebrate host stages (amastigotes, black bar) for L. donovani, L. mexicana, and T. cruzi. Equivalent amounts of protein from lysed parasites were measured for OPB activity at pH 8.0 using the Z-PR-AMC substrate.

FIGURE 2.

Purification scheme and identification of OPB from L. donovani lysate. The soluble fraction of L. donovani promastigote lysate was fractionated by Q-Sepharose ion exchange chromatography. OPB-containing fractions were identified by testing for cleavage of Z-PR-AMC at pH 8.0. Activity was plotted against fraction number, and a single peak of activity was observed. The maximally active fraction, fraction 60, was resolved on an SDS-polyacrylamide gel. Major bands were excised from the gel, and the proteins were identified by tandem mass spectrometry. The protein contained in the excised gel from 75 to 90 kDa was identified as OPB. OPB has a predicted molecular mass of 83.1 kDa.

FIGURE 3.

Determination of the pH preference of OPB. Purified OPB was tested for pH preference by measuring the hydrolysis of Z-PR-AMC in different pH buffers. Activity was compared at pH values 3.0–10.5 with a 0.5-pH unit step size in 100 mm citrate phosphate buffer (black diamonds; pH 3.0–7.0), sodium phosphate buffer (white squares; pH 6.5–8.0), Tris-HCl buffer (black triangles; pH 7.5–9.0), and glycine-NaOH buffer (white circles; pH 8.5–10.5). Error bars, S.D.

FIGURE 4.

Tetrapeptide substrate specificity profiling of OPB using a positional scanning synthetic combinatorial library. Subsite preference was determined using P1–P4 combinatorial libraries. All of the substrates contained an ACC fluorogenic leaving group. Assays were performed in triplicate, and the mean ± S.D. (error bars) is shown. Activity levels were normalized to the maximum mean value observed for each library. The x axis indicates the amino acid held constant at each position for a given library, designated by the one-letter code (with n representing norleucine). The determined preference for each subsite is in boldface type below the x axis (X indicates that multiple amino acids are permissible in the given subsite).

FIGURE 5.

Genetic and enzymatic analysis of OPB knockouts. A, successful deletion of both genomic copies of the OPB gene were determined by Southern blot analysis. Digested genomic DNA from wild type, single copy, and double copy knockouts were analyzed at the OPB locus for the presence of either the wild type locus or the knock-out cassette. The blot was stripped and reprobed for the OPB gene itself to ensure that the gene was not relocated to another site in the genome. B, lysates from wild type, single copy, and double copy knockouts were analyzed for OPB enzymatic activity using the Z-PR-AMC substrate at pH 8.0.

FIGURE 6.

Proteomic analysis of OPB knock-out parasites. A, two-dimensional gel electrophoresis was used to compare lysates from wild type and OPB double knock-out parasites. The arrows indicate four spots that were significantly more intense in lysate from the knock-out parasites. The intensity increase was observed on replicate gels. The proteins in these spots were identified by mass spectrometry and were all found to be enolase. B, to confirm that the accumulated isoforms of enolase found in the OPB knock-out parasites did not represent increased glycolytic enolase, the glycolytic pathway function of enolase was analyzed by measuring the conversion of 2-phosphoglycerate to phosphoenolpyruvate spectrophotometrically. Error bars, S.D. of three separate experiments.

FIGURE 7.

Differential localization of enolase in OPB knock-out versus wild type Leishmania. Wild type and OPB(−/−) Leishmania were fractionated by differential lysis. Western blots for enolase were performed on these cell extracts. Cell fractions were as follows: cell surface fraction (A), cytoplasmic fraction (B), nuclear fraction (C), and cytoskeletal fraction (D). Equivalent levels of enolase were detected in the cytoplasmic and nuclear fractions of both wild type and OBB −/− parasite, and it was not detected in the cytoskeletal fractions. Enolase was found to have accumulated on the cell surface of OPB(−/−) Leishmania but was not present on the cell surface of the wild type parasites.

FIGURE 8.

Gene expression of macrophages infected by OPB knock-out parasites. Murine bone marrow-derived macrophages were infected with either wild type or OPB(−/−) parasites for 0, 2, 6, 12, and 24 h. Gene expression from these macrophages was analyzed by microarray. A and B, array data for wild type (A) and OPB(−/−) (B) infections were analyzed to find statistically significant changes in gene expression compared with uninfected macrophages using a false discovery rate threshold of <1.0% and a minimum -fold change of 2. The x axis indicates the expression level of a given gene in the uninfected control macrophages. The y axis indicates the expression level observed in the infected macrophages. Genes with a significant change in expression after Leishmania infection are colored red for up-regulation and green for down-regulation. In the wild type infections, only 23 genes were differentially expressed (false discovery rate = 0.00%) compared with uninfected cells; however, in the OPB(−/−) infections, there were 459 significantly up-regulated genes (false discovery rate = 0.67%). C, heat maps of the array data were clustered to illustrate the differences between the different time courses. The OPB knock-out-infected macrophage arrays show a significant increase in gene expression (red) compared with the uninfected and wild type Leishmania-infected macrophage arrays.

FIGURE 9.

Mouse infections by wild type and OPB(−/−) L. major. BALB/c mice (n = 5) were infected subcutaneously in the left hind footpads with wild type (solid circle) or knock-out (empty circle) L. major. Footpad swelling was measured weekly, and the footpad thickness was plotted over time. Error bars indicate the S.D. between the five mice in each group. The mice were sacrificed at 180 days postinfection. No swelling was observed in the right hind (uninfected) footpads. Significant footpad swelling occurred at 46 days PI in wild type-infected mice (p = 0.002) and 116 days postinfection in OPB knock-out-infected mice (p = 0.036).

FIGURE 10.

Model for OPB function in the Leishmania life cycle. This schematic details the proposed function of Leishmania OPB during infection of a vertebrate host. In a wild type infection, surface enolase (black diamonds) binds host plasminogen (dark gray circles) on the parasite cell surface. As the parasite begins differentiating into an amastigote, OPB is up-regulated, thereby clearing surface enolase and plasminogen. The amastigotes replicate undetected within the macrophage. In an OPB(−/−) infection, when the parasite differentiates into the amastigote stage, enolase and plasminogen are retained on the cell surface. This infection is detected by the macrophage, resulting in increased macrophage gene transcription and reduced parasite virulence.