A potential role for ICP, a Leishmanial inhibitor of cysteine peptidases, in the interaction between host and parasite

Abstract

The biological role of a natural inhibitor of cysteine peptidases (designated ICP) of Leishmania has been investigated by genetic manipulation of the parasite. Null mutants grew normally in vitro, were as infective to macrophages in vitro as wild-type parasites, but had reduced infectivity to mice. Mutants re-expressing ICP from a single gene gave partial restoration of virulence in vivo, whereas mutants overexpressing ICP secreted the inhibitor and showed markedly reduced virulence in mice. Promastigotes of the null mutants had similar cysteine peptidase activities as the wild-type parasites, suggesting that ICP is not required for the expression or processing of the enzymes. The only proteins found to bind to ICP in promastigote cell lysates were fully processed forms of CPA and CPB, showing that ICP does not bind in abundance either to zymogens of the cysteine peptidases or other leishmanial proteins. However, only a small proportion of ICP colocalized with CPA and CPB in the promastigote (in the endoplasmic reticulum and Golgi) and the majority of ICP resided in vesicles that are apparently distinct from endosomes and the multivesicular tubule (MVT)-lysosome. These data suggest that ICP has a role other than modulation of the activity of the parasite's own cysteine peptidases and their normal trafficking to the MVT-lysosome via the flagellar pocket. The finding that ICP partially colocalized with an endocytosed cysteine peptidase leads us to postulate that ICP has a role in protection of the parasite against the hydrolytic environment of the sandfly gut and/or the parasitophorous vacuole of host macrophages.

Fig. 1. Targeted replacement of the ICP gene

A. Schematic representation of the ICP locus and the plasmid constructs used for gene replacement, re-integration and over-expression. ORFs are shown as arrows, intergenic and flanking DNA sequences are shown as boxes. Restriction enzymes used for the different constructs are shown (see text), as well as the expected sizes of the AgeI/NaeI fragments revealed by the Southern blot. DHFR, dihydrofolate reductase gene; BLA, blasticidin resistance gene; HYG, hygromycin resistance gene; NEO, neomycin resistance gene, PAC, puromycin resistance gene. B. Southern blot analysis. Genomic DNA was digested with AgeI and NaeI, separated on a 1% agarose gel, blotted onto a nylon membrane and hybridised with ³²P-labelled DNA probes. The hybridisation probes were fragments containing the entire coding region of their respective genes. lane 1, wild-type L. mexicana; lane 2, Δicp ; lane 3, Δicp:: P_RRNA​ ICP; lane 4, Δicp[pXG ICP]. The positions of the molecular size markers are shown on the right.

Fig. 2. Protein expression

A. Whole-cell lysates prepared from stationary-phase promastigotes of wild-type (WT), Δicp, Δicp:: P_RRNAICP and Δicp[pXG ICP] were analysed using 12% (w/v) SDS-PAGE and immunoblotted with affinity-purified polyclonal antibodies raised against ICP, and monoclonal antiserum raised against gp63 to confirm equivalent protein loading. The equivalent of approximately 10⁷ parasites was loaded per lane, molecular masses are indicated on the right. B. Proteins were isolated from spent medium from cultures containing ∼10⁸ promastigotes or axenic amastigotes of wild-type (WT), Δicp, and Δicp[pXG ICP] parasites. Proteins were analysed by Western blot using affinity-purified anti-ICP antibody. C. Immunoprecipitation of ICP and its associated proteins from L. mexicana promastigote lysates. The affinity-purified anti-ICP antibodies were coupled to a protein A/G Sepharose column and used to purify protein complexes from stationary-phase promastigotes lysates. Approximately 5 ×10⁹ cells were used from wild-type Leishmania. Proteins identified by mass spectrometry are indicated with arrows. Molecular masses are shown on the left. D. Analysis of CPA and CPB. Lysates of 10⁷ stationary phase promastigotes were used for gelatin SDS-PAGE to detect CPB activities (upper panel) and Western blotting using anti-CPA antibody (lower panel). Molecular masses are indicated on the left. Asterisks and arrows mark the positions of precursor CPB and mature CPB, respectively.

Fig. 3. ICP null mutants are infective to macrophages and mice

A. In vitro macrophage infectivity assays. Wild-type (WT) and transgenic stationary-phase L. mexicana promastigotes (P), and axenic amastigotes (A), were incubated with mouse peritoneal macrophages at a ratio of 5:1 and the parasite load determined by counting the number of infected macrophages after 7 days of incubation. The data show the percentage of infected macrophages ± SD from triplicate infections. B. Infection of BALB/c mice with wild-type and transgenic stationary-phase L. mexicana promastigotes. Mice were challenged with 5 × 10⁵ in the left hind footpad. The swelling caused by the respective cell lines was recorded. Data shown (mean lesion diameter ± SD from groups of 5 mice) are representative of three experiments done with two independent clones for each line. C. Parasite loads in footpad lesions from infected BALB/c mice. Footpad lesions were dissected at the end of the infection experiment (week 12), tissue was disrupted in PBS and the number of parasites was estimated microscopically by counting.

Fig. 4. Localisation of ICP, CPA and CPB

A. Immunofluorescence analysis of early stationary-phase promastigotes with purified rabbit anti-ICP antibodies (red) and rabbit anti-CPA or anti-CPB antibodies (green). B. Immunofluorescence analysis of stationary-phase promastigotes with purified rabbit anti-CPB antibodies (red) and anti-CPA antibodies (green). Primary antibodies were labelled directly with the desired fluorophore using the Zenon labelling kit (Molecular Probes) and the fluorescence images have been deconvolved using the Volume Deconvolution module of the Openlab Software (Improvision). . Merged images are magnified, with DAPI-stained DNA (blue) of the nucleus (n) and the kinetoplast (k), the scale bar represents 10 μm. The co-localised signal is yellow, highlighted by arrows in A.

Fig. 5. Co-localisation with endocytosed markers

A. Promastigotes were exposed to FITC-conjugated Con A (green) at 4° C for 10 min prior to a chase for 2 h at 25° C to permit accumulation in the flagellar pocket (Fp, arrowed) and late endosomes (L, arrowed). Cells were fixed and stained with anti-CPB or anti-ICP rabbit antibodies, followed by Texas Red-conjugated goat anti-rabbit antibody (red). B. FITC-conjugated tomato lectin (TL) was added to promastigotes at 4° C for 10 min prior to a chase for 30 min at the same temperature to permit accumulation in a specific compartment (green). Cells were fixed and stained with anti-CPB or anti-ICP rabbit antibodies, followed by Texas Red-conjugated goat anti-rabbit antibody (red). Merged images are magnified, with DAPI-stained DNA (blue) and co-localised signal, where present, is yellow. The scale bar represents 10 μm.

Fig. 6. Co-localisation with ER and Golgi markers

A. Early stationary-phase promastigotes were treated for microscopy and stained with mouse anti-LmLCB2 (an ER protein), followed by a goat anti-mouse FITC-conjugate antibody (green), and with anti-CPB or anti-ICP rabbit antibodies, followed by a goat anti-rabbit Texas Red-conjugate antibody (red). Merged images are shown with DAPI-stained DNA (blue) of the nucleus (n) and the kinetoplast (k), the scale bar represents 10 μm. The co-localised signal is yellow, highlighted by arrows. B. Log-phase promastigotes expressing GFP-fused GRIP protein (trans-Golgi resident protein) were fixed and stained with mouse anti-GFP antibody, followed by a goat anti-mouse FITC-conjugate antibody (green), and with anti-CPB or anti-ICP rabbit antibodies, followed by Texas Red-conjugated goat anti-rabbit antibody (red). Merged images are magnified, with DAPI-stained DNA (blue) and co-localised signal is yellow. The scale bar represents 10 μm.

Fig. 7. Internalisation of papain

Log phase promastigotes were incubated for 10 mins with 1 μg of papain previously treated with sulfo-NHS-SS-biotin. Surface biotin was removed by reducing the disulphide bonds using glutathione. Cells were treated for fluorescence and internal papain was revealed by using Alexa Fluor 594-conjugated streptavidin (red). Cells were also stained with anti-ICP antibodies, followed by FITC-conjugated goat anti-rabbit antibody (green). The fluorescence images have been deconvolved using the Volume Deconvolution module of the Openlab Software (Improvision). Merged images are magnified, with DAPI-stained DNA (blue) of the nucleus and the kinetoplast. Co-localised signal is yellow and some is highlighted with arrows. The scale bar represents 10 μm.