Absence of metalloprotease GP63 alters the protein content of Leishmania exosomes

Abstract

Protozoan parasites of Leishmania genus are able to successfully infect their host macrophage due to multiple virulence strategies that result in its deactivation. Recent studies suggest Leishmania GP63 to be a critical virulence factor in modulation of many macrophage molecules, including protein tyrosine phosphatases (PTPs) and transcription factors (TFs). Additionally, we and others recently reported that Leishmania-released exosomes can participate in pathogenesis. Exosomes are 40-100 nm vesicles that are freed by many eukaryotic cells. To better understand the GP63-dependent immune modulation of the macrophage by Leishmania parasites and their exosomes, we compared the immunomodulatory properties of Leishmania major (WT) and L. major gp63-/- (KO) as well as their exosomes in vitro and in vivo. Importantly, we observed that Leishmania exosomes can modulate macrophage PTPs and TFs in a GP63-dependent manner. In addition, our qRT-PCR analyses showed that WT parasites were able to downregulate multiple genes involved in the immune response, especially cytokines and pattern recognition receptors. KO parasites showed a strongly reduced modulatory capacity compared to WT parasites. Furthermore, comparison of WT versus KO exosomes also showed divergences in alteration of gene expression, especially of chemokine receptors. In parallel, studying the in vivo inflammatory recruitment using a murine air pouch model, we found that exosomes have stronger proinflammatory properties than parasites and preferentially induce the recruitment of neutrophils. Finally, comparative proteomics of WT and KO exosomes surprisingly revealed major differences in their protein content, suggesting a role for GP63 in Leishmania exosomal protein sorting. Collectively our data clearly establish the crucial role of GP63 in dampening the innate inflammatory response during early Leishmania infection, and also provides new insights in regard to the role and biology of exosomes in Leishmania host-parasite interactions.

Figure 1. Purification of exosomes from CM of WT and KO parasites.

A and B. Silver stainings of sucrose density gradient fractions of WT and KO exosomes respectively. Enrichment of bands can be observed in fractions 8, 9 and 10, which correspond to the density of exosomes. C. Western blotting of sucrose density gradient fractions of WT exosomes against GP63. Again accumulation of GP63 can be observed at fractions 8, 9 and 10. D and E. Transmission electron microscopy of WT and KO exosomes shows that WT and KO exosomes have similar size and morphology. Arrows point to exosomes. F. Gelatin zymogram of 3 µg of exosomes and lysates shows that GP63 remains active after exosome purification. All results are representatives of at least 3 independent experiments.

Figure 2. WT but not KO exosomes cleave macrophage PTPs dose-dependently.

A. Western blotting shows that only WT but not KO parasites and exosomes can induce cleavage of prominent macrophage PTPs, TC-PTP, PTP-1B and SHP-1 after 3 h of incubation or infection. B. In gel PTP assay shows multiple modulations in the PTP profile of the macrophage following WT infection or incubation with WT exosomes, suggesting that cleavage fragments are enzymatically active. These modulations do not occur with KO parasites or with exosomes. All results are representatives of at least 3 independent experiments.

Figure 3. Modulation of TFs by Leishmania parasites and exosomes.

EMSAs show that Leishmania infection can result in translocation of a modified form of NF-κB (A) and AP-1 (B) degradation as previously reported. Stimulation with parasite exosomes shows that KO exosomes induce stronger translocation of NF-κB and AP-1 into the nucleus and thus appear more inflammatory compared to WT exosomes. Results are representatives of at least 3 independent experiments. C.S. Specific Competitor, 100× concentration of non-labelled oligo. C.N. Non-Specific competitor, 100× concentration of non-labelled consensus SP-1 oligo. N.S. Non-specific.

Figure 4. Gene ontology analyses of upregulated and downregulated genes.

WT parasites, and to some extent KO parasites strongly downregulate expression of many immune-related genes, especially with plasma membrane, extracellular associated and nucleus GO terms, indicating secretory factors, receptors and transcription regulation proteins. Although WT exosomes do inhibit expression of certain genes, exosomes in general have a more stimulatory nature.

Figure 5. Recruitment of inflammatory cells to the air pouch in response to Leishmania parasites and exosomes.

A, B and C show recruitment of neutrophils, monocytes/macrophages and eosinophils/mast cells calculated based on differential count respectively. D shows Log₁₀ of the neutrophil/macrophage ratio, illustrating stronger recruitment of macrophages by parasites, compared to exosomes. Results show average of 3 separate experiments plus SEM. Statistical significance is measured against PBS-injected mice unless specified with a line (*: P-value <0.05, **: P-value <0.001, ***: P-value <0.0001).

Figure 6. Proteomic analysis of WT and KO exosomes.

A Venn diagram of proteins shared between WT and KO exosomes as well as unique proteins. B Venn diagram of common proteins between WT and KO exosomes, showing number of proteins with higher abundance (2×).

Figure 7. RSC exosomes show an intermediated phenotype between WT and KO in terms of presence and abundance of proteins.

Proteins found in exosomes have been categorized as present (red) or absent (blue) in WT exosomes. Considering presence and absence of proteins, and also having similar abundance to WT, compared with KO, RSC exosomes show an intermediate phenotype between WT and KO exosomes. Refer to the results section for more detailed description.

Figure 8. Verification of the proteomic data.

A. Silver staining of 2 µg of exosomes and lysates shows that despite similarity of lysate band patterns, WT and RSC exosome band patterns differ from that of KO exosomes. B. Western blotting also shows equal levels of protein in parasite lysates, except for GP63 that is absent in KO. However, levels of tubulin and LACK protein appear to change in KO exosomes and go back to their WT levels in RSC. HSP83 levels appear to be unaffected. All results are representatives of at least 3 independent experiments.