Oligopeptidase B deficient mutants of Leishmania major

Abstract

Oligopeptidase B is a clan SC, family S9 serine peptidase found in gram positive bacteria, plants and trypanosomatids. Evidence suggests it is a virulence factor and thus therapeutic target in both Trypanosoma cruzi and T. brucei, but little is known about its function in Leishmania. In this study L. major OPB-deficient mutants (Δopb) were created. These grew normally as promastigotes, had a small deficiency in their ability to undergo differentiation to metacyclic promastigotes, were significantly less able to infect and survive within macrophages in vitro, but were virulent to mice. These data suggest that L. major OPB itself is not an important virulence factor, indicating functional differences between trypanosomes and Leishmania in their interaction with the mammalian host. The possibility that an OPB-like enzyme (designated OPB2) in L. major might compensate for the loss of OPB in Δopb was investigated via by mapping its sequence onto the 1.6Å structure of L. major OPB. This suggested that the residues involved in the S1 and S2 subsites of OPB2 are identical to OPB and hence the substrate specificity would be similar. Consequently there may be redundancy between the two enzymes.

Fig. 1

Expression of OPB in L. major Western immunoblot showing the expression of OPB in the three life cycle stages of L. major. Lane 1 = procyclic promastigotes, 2 = metacyclic promastigotes, 3 = amastigotes. Purified anti-OPB antibody was used, with anti-T. brucei elongation factor 1α (EF-1α) antibody as a loading control. A lysate of 5 × 10⁶ cells was run per lane.

Fig. 2

Deletion of L. major OPB. A: Schematic representation of the OPB locus and the plasmid constructs used for gene replacement. The OPB and antibiotic resistance genes are shown as arrows, flanking DNA sequences are shown as boxes. XhoI fragments are shown. 5′-DHFR and 3′-DHFR, dihydrofolate reductase flanking regions; HYG, hygromycin resistance gene and SAT, streptothricin acetyltransferase resistance gene. B: Southern blot analysis of genomic DNA digested with XhoI, separated on a 0.7% agarose gel, blotted onto a nylon membrane and hybridized using DNA probes corresponding to a 300 bp fragment of OPB, and the HYG and SAT genes. 1; L. major WT, 2; heterozygote, 3; Δopb clone 10, 4; Δopb clone 21, 5; Δopb clone 10 re-expressing OPB and 6; Δopb clone 21 re-expressing OPB. Molecular size markers are shown on the left. C: Demonstration of OPB protein levels in L. major WT promastigotes and Δopb by western immunoblot. Lanes as in B. Purified anti-OPB antibody was used and EF-1α antibody as a loading control. A lysate of 5 × 10⁶ cells was run in each lane. D: Demonstration of over-expression of OPB in L. major cells by western immunoblot. Lane 1; L. major WT, 2; WT[OPB] population, 3; L. major WT, 4; WT[pNUS] population and 5; WT[OPB^S577G] population. Purified anti-OPB antibody was used, with anti-EF-1α antibody as a loading control. A lysate of 5 × 10⁶ cells was run in each lane.

Fig. 3

Analysis of infectivity and metacyclogenesis of Δopb clones and re-expressing cell lines. A: Western immunoblot to compare HASPB levels. Lane 1; L. major WT cells, 2; Δopb clone 10, 3; Δopb clone 10 re-expressing OPB. Anti-HASPB antibody was used, with anti-EF-1α antibody as a loading control. A lysate of 5 × 10⁶ cells was run per lane. B: Percentage of infected peritoneal exudate macrophages (PEM). PEM were infected at a ratio of 6 metacyclic promastigotes to 1 macrophage and were incubated for between 24 hours and 6 days, with non-phagocytosed Leishmania washed off after 24 hours. Each value is the mean of 6-8 replicates over two experiments ± SEM. *= p <0.001, in comparison to wild type. C: Number of amastigotes per 100 PEM. PEM were infected at a ratio of 6 metacyclic promastigotes to 1 macrophage and were incubated for between 24 hours and 6 days, with non-phagocytosed Leishmania washed off after 24 hours. Each value is the mean of 6-8 replicates over two experiments ± SEM. D: Growth of footpad lesions in BALB/c mice. Footpads infected with Δopb clone 10 were compared to footpads infected with WT L. major. 1 × 10⁵ metacyclic promastigotes were injected per footpad. Each value is the mean of 6 mice ± SEM.

Fig. 4

Reduction of serine peptidase activity in Δopb A: Effect of deletion and re-expression of OPB in L. major promastigotes on the cleavage of 5 μM Bz-R-AMC. Relative activity was calculated compared to WT for each experiment. For each assay, 6 × 10⁶ cells were used, with the mean of between 5 and 11 experiments shown ± SEM. B: Effect of over-expression of OPB in L. major promastigotes on the cleavage of 5 μM Bz-R-AMC. Relative activity was calculated compared to WT for each experiment. For each assay, 6 × 10⁶ cells were used, with the mean of between 4 and 11 experiments shown ± SEM. C: Fractionation of OPB. Western immunoblot showing partition of OPB to either soluble or membrane fraction of L. major. Lane 1; soluble fraction, 2; pelleted fraction. Purified anti-OPB antibody was used, with controls of anti-EF-1α and anti-GP63 antibodies.

Fig. 5

Sequence alignment of L. major OPB and OPB2. Alignment based on a multiple sequence alignment using eight OPB homologues performed by CLUSTALW. The catalytic domain of OPB is underlined in red and the propeller domain left blank. Predicted inserts of OPB2 into the OPB sequence are shown in pink. Identical residues are shaded, with those involved in the catalytic triad in red. Red, green and blue stars highlight all of the residues involved in the S1-S3 binding pockets, respectively. Solid stars represent hydrogen-bonding interactions while the hollow stars denote pi-cation or hydrophobic interactions. This shows that all of the important residues involved in the binding of antipain in OPB are conserved in OPB2.