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. Author manuscript; available in PMC: 2015 Nov 14.
Published in final edited form as: Cell Rep. 2015 Oct 22;13(5):957–967. doi: 10.1016/j.celrep.2015.09.058

Exosome secretion by the parasitic protozoan Leishmania within the sand fly midgut

Vanessa Diniz Atayde 1,2, Hamide Aslan Suau 3,4, Shannon Townsend 3, Kasra Hassani 1, Shaden Kamhawi 3,*, Martin Olivier 1,2,*
PMCID: PMC4644496  NIHMSID: NIHMS726141  PMID: 26565909

SUMMARY

Despite several studies describing the secretion of exosomes by Leishmania in vitro, observation of their formation and release in vivo has remained a major challenge. Herein, we show that Leishmania constitutively secretes exosomes within the lumen of the sand fly midgut through a mechanism homologous to the mammalian pathway. Through egestion experiments, we demonstrate that Leishmania exosomes are part of the sand fly inoculum and are co-egested with the parasite during the insect’s bite possibly influencing the host infectious process. Indeed, co-inoculation of mice footpads with L. major plus midgut-isolated or in vitro-isolated L. major exosomes resulted in a significant increase in footpad swelling. Notably, co-injections produced exacerbated lesions through overinduction of inflammatory cytokines, in particular IL-17a. Our data indicate that Leishmania exosomes are an integral part of the parasite’s infectious life cycle and propose to add these vesicles to the repertoire of virulence factors associated to vector-transmitted infections.

INTRODUCTION

Exosomes have been the focus of numerous studies due to their involvement in intercellular communications, especially amongst immune and tumor cells. They are secreted by most eukaryotic cell types and comprise a variety of functions due to their variable content, which often mirrors the secreting cell. In the classical pathway, exosomes are formed via the invagination of endocytic compartments generating multivesicular bodies (MVBs), and are released to the extracellular space after fusion of the MVBs with the plasma membrane (Bobrie et al., 2011; Bobrie and Thery, 2013). Despite the abundant knowledge obtained through studies using exosomes purified in vitro or from various biological fluids (Taylor and Gercel-Taylor, 2013), observation of their formation and release in vivo remains a major challenge.

In vitro-isolated exosomes play a crucial role in host-pathogen interactions. Vesicles secreted by infected cells contain substantial amounts of pathogen molecules, which are sufficient to induce modifications in non-infected neighboring cells or act as antigen presenters for the immune system. This is the case for Epstein-Barr virus- (Koppers-Lalic et al., 2014), Mycobacterium- (Bhatnagar and Schorey, 2007), Toxoplasma gondii- (Bhatnagar et al., 2007) and Leishmania-infected cells (Hassani and Olivier, 2013), among others. Correspondingly, studies have granted biological importance to exosomes secreted directly by pathogens (Hassani et al., 2011; Hassani et al., 2014; Silverman and Reiner, 2011a).

Leishmania spp. are obligate intracellular parasites which primarily infect macrophages in the mammalian host and are transmitted through regurgitation during the bite of an infected sand fly. They are phylogenetically considered as ancient eukaryotes (Hedges et al., 2004; Sibley, 2011) and are shown to secrete bona fide exosomes in vitro. Experiments performed with mice and macrophages have shown that these exosomes possess immunomodulatory and signaling-inducing activities, corroborating the presence of parasite virulence factors in their content such as the surface metalloprotease GP63 (Hassani et al., 2011; Hassani et al., 2014; Silverman et al., 2010b; Silverman and Reiner, 2011a). Thus, it is postulated that these vesicles contribute to the multitude of factors determining the form and severity of the leishmaniases, a spectrum of diseases that ranges from self-healing cutaneous to fatal visceral forms and represents a major public health problem worldwide (www.who.int/leishmaniasis).

To date, there is no direct evidence supporting the secretion of exosomes by Leishmania in vivo, either within the sand fly vector or within the mammalian host. Here we demonstrate the formation and release of exosomes by Leishmania residing in the midgut of its natural vector, the sand fly. Moreover, we fully characterize exosomes isolated from infected sand fly midguts, and compare them to previously described in vitro-isolated Leishmania exosomes. Ultimately, we demonstrate that in vivo-secreted Leishmania exosomes are egested by the sand fly alongside parasites during the bite and address the potential role of these vesicles in the establishment and pathology of cutaneous leishmaniasis.

RESULTS

Leishmania constitutively secretes exosome-like vesicles into the lumen of the sand fly midgut

Outside the mammalian host, Leishmania is confined to the digestive tract of sand flies. In this environment, the parasite differentiates into multiple forms as they progress towards the anterior midgut where non-replicative infective metacyclic promastigotes accumulate behind the stomodeal valve, positioned for ready transmission to the mammalian host during the sand fly’s blood meal (Kamhawi, 2006). To establish whether Leishmania releases exosomes in vivo during its developmental cycle in the sand fly, we dissected Lutzomyia longipalpis midguts infected with L. infantum, its natural parasite, or with L. major for TEM analysis. L. longipalpis is not L. major’s natural vector, but sustains its development and transmission. Midguts infected with L. infantum or L. major showed microvillar structures and several Leishmania in the lumen (Fig. S1C–F and S1G–L, respectively). Uninfected midguts presented similar organization, except for the absence of parasites (Fig. S1A and S1B). For L. major-infected sand flies, we isolated anterior midguts in order to observe infective promastigotes. TEM showed increased density of parasites in this region (Fig. S1M–Q). Remarkably, a total of 64.3% of L. infantum promastigotes, 72.5% of L. major promastigotes and 72.7% of L. major promastigotes in the anterior midgut were captured secreting single or several vesicles from membrane surfaces (Fig. 1A, 1B, 1E, 1F, S2A and S2B). These vesicles were rounded or cup-shaped, limited by a membrane bilayer and around 50 to 120 nm in diameter, which are characteristics of exosomes. In addition, we often observed clusters of exosome-like vesicles in the lumen (Fig. S1F, S1L and S1R). Lastly, we captured a few vesicles that appeared to derive from midgut cells due to their proximity to microvillar structures (Fig. S1C and S1K).

Figure 1. Leishmania constitutively releases vesicles in the lumen of the sand fly midgut.

Figure 1

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L. infantum- and L. major-infected midguts were processed for TEM. (A, E, F) Promastigotes secreting single membrane vesicles (red arrows, insets). (B, F) Promastigotes secreting several membrane vesicles (red arrows, inset). (C, G) MVBs fused to the parasite’s membrane, releasing vesicles (red arrows) into the midgut. (D, H) Flagellar pockets containing vesicles (green arrows). (D) Vesicles released into the sand fly midgut (red arrows). The images are representative of 3 independent experiments. K, kinetoplast, N, nucleus, F, flagellum, FP, flagellar pocket, MV, microvillar structures, MVB, multivesicular bodies. See also Figures S1, S2 and S3.

Sand fly-residing Leishmania secretes exosome-like vesicles from MVBs and flagellar pockets

Observing both L. infantum and L. major cells in vivo by TEM, we found a number of cytoplasmic MVB-like compartments, which in several cases were approaching the parasite’s membrane (Fig. S3A–D, S3G–I, S3M and S3N) or releasing their vesicle content to the sand fly midgut (Fig. 1C, 1G, S2C, S3E, S3F, S3J–L, S3O and S3P). These findings suggested that the mechanism of Leishmania vesicle secretion is somewhat homologous to the classical exosome pathway described for higher eukaryotes. Additionally, we observed several exosome-like vesicles accumulated in flagellar pockets or exiting the parasite through the flagellar pocket (Fig. 1D, 1H, S2D), an event previously reported in vitro (Silverman et al., 2008). We also captured regions of fusion between MVBs and flagellar pockets (Fig. S2D and S3A).

In vivo- and in vitro-secreted Leishmania exosomes are exceptionally similar

To confirm that vesicles released by sand fly-residing Leishmania were authentic exosomes in respect to previously characterized in vitro-isolated vesicles, we isolated and fully characterized them by TEM, mass spectrometry (MS) and western blot. For these purposes, infected and uninfected sand fly midguts were dissected along their longitudinal surface and their content was collected in a fresh drop of PBS (Fig. S4A), from where vesicles were collected. Observed by negative staining, infected midgut-isolated vesicles were identical in appearance to in vitro-isolated Leishmania exosomes (Fig. 2A–C and S4B–D). Fewer vesicles, more heterogeneous in size and shape were isolated from uninfected midguts.

Figure 2. The proteome of in vivo-secreted Leishmania vesicles.

Figure 2

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Midgut lavages were separated from the infected pellet (IMP) and the uninfected pellet (UMP). Vesicles were isolated from the supernatants (infected midgut vesicles, IMV; uninfected midgut vesicles, UMV). (A) Midgut-isolated L. infantum vesicles. (B) Midgut-isolated L. major vesicles. (C) In vitro-isolated L. major exosomes. (D) Distribution of the proteins from midgut-isolated L. infantum vesicles identified by databases (db). (E) Peptide fold-change for proteins belonging to IMV (LIME) compared to IMP. (F) The LIME interaction network. Clusters: I. Antioxidant and metabolic proteins. II. Chaperones and proteasome proteins. III. Vesicular trafficking and microtubule-associated proteins. IV. Translation proteins. (G) Western blots of: UMP and L. infantum (L.i.) IMP; uninfected and L.i-infected midgut lavages before (UML, IML) or after vesicle purification (UMV, IMV); in vitro-purified L. major (L.m.) exosomes (Exo) and L.m. whole cell lysates (Pro); L.m. IMVs (2 experiments). The results are representative of 3 independent experiments. See also Figures S4, S5 and S6.

Next, we compared the protein content of infected and uninfected midgut vesicles (IMV and UMV) by MS. In the experiments with L. infantum-infected sand flies, 143 proteins were identified by searching the L. infantum database. From those, 124 were unique to IMV, while 19 were shared between IMV and UMV (Fig. 2D). The majority of the shared proteins (Table S1, top, 125–143) were highly conserved between L. infantum and L. longipalpis, such as actin (71% identity) and glyceraldehyde 3-phosphate dehydrogenase (53.6% identity). Although we could not quantify the exact percentage of sand fly-derived material in IMV, all shared proteins were previously found in Leishmania exosomes and for this reason were kept in the final L. infantum Midgut Exoproteome (LIME) list (Table S1, top). Searching the L. longipalpis database, 52 proteins were identified, half of them digestive tract-related (Table S1, bottom). In contrast to the results with the Leishmania database, 50 out of 52 proteins were shared between IMV and UMV and only 2 proteins were unique to UMV (Fig. 2D). We obtained similar results in the experiments with L. major-infected sand flies (Fig S5A), from where we generated the L. major Midgut Exoproteome (LMME) list (Table S2, top). When comparing LMME content to LIME, 24 out of 56 proteins were common, which included several exosome markers such as GP63 and HSP70 (Tables S1, top and S2, top).

Using proteomic tools for further characterization, only 15 out of the 143 LIME proteins had a predicted signal peptide, while 58 proteins were predicted to be secreted unconventionally -a feature of exosomal proteins (Hassani et al., 2011; Thery et al., 2009). On the other hand, from the 52 proteins identified with the sand fly database, 25 had predicted signal peptides and only 6 were predicted as unconventionally secreted (Table S1). Similar results were obtained for LMME (Table S2). Compared with previous works with in vitro-purified Leishmania vesicles (Hassani et al., 2011; Santarem et al., 2013; Silverman et al., 2010a), the majority of the LIME identified proteins (99 out of 143) had identical matches in at least one of the 3 databases (Table S1, top). Likewise, 49 out of 56 LMME proteins had been previously detected in Leishmania exosomes (Table S2, top).

Lastly, we verified if in vivo Leishmania exosomes, as observed in vitro, presented distinct protein profiles when compared to the cells from which they were collected. Comparing the proteome of IMVs with that of infected midgut pellets (IMP) containing promastigotes, 47 proteins were enriched in LIME and 27 in LMME by 2-fold or more (Fig. 2E and S5B). A number of these proteins have been described as virulence and/or immunogenic factors including GP63 (Olivier et al., 2012), calpain-like cysteine peptidases (Mottram et al., 2004), HSP70 (Rafati et al., 2007), tryparedoxin peroxidase (Iyer et al., 2008) and surface antigen protein (Kemp et al., 1998) (Table S3). Gene ontology revealed that in general IMVs mirrored the IMP physiological state (Fig. S6). To assess the functional characteristics of the exoproteomes, we generated protein-protein interaction networks (Fig. 2F and 5SC). The complete list of proteins mapped in the interaction networks is provided in Table S4. Western blots confirmed the presence of GP63 in IMVs. In contrast, HSP83, which is not enriched in exosomes and glycosomal HGPRT, a control for parasite contamination in exosome preparations, were detected only in IMP (Fig. 2G). We obtained similar results when we compared L. major parasites with in vitro-purified L. major exosomes (Fig. 2G).

Collectively, our data defines midgut-isolated Leishmania vesicles as bona fide exosomes and place these vesicles in the parasite’s natural life cycle. In addition, their location in the lumen, especially in the anterior midgut, suggests that midgut exosomes are co-egested alongside parasites during the bite of an infected sand fly, similarly to what has been reported for the promastigote secretory gel, an exacerbating factor of Leishmania infection (Rogers et al., 2009; Rogers et al., 2004). We believe that Leishmania exosomes egested alongside parasites exert their own influence on the infectious process perhaps by modulating innate immune responses during early events of the infection, as described for these vesicles in vitro (Hassani et al., 2011; Hassani et al., 2014).

Leishmania exosomes are egested during the sand fly bite

To answer whether Leishmania exosomes, copiously secreted within the sand fly midgut (including the anterior midgut) were present in the sand fly inoculum, L. infantum- and L. major-infected sand flies where fed in vitro on RPMI medium or vesicle-free plasma through a chicken-skin membrane as previously described (Rogers et al., 2004). Sand fly egests where collected and used for exosome isolation. Excitingly, TEM images showed exosomes in both L. infantum and L. major-infected inocula (Fig. 3A and S7A). A few exosomes and smaller vesicles were also isolated from uninfected inocula (Fig. S7A).

Figure 3. The sand fly inoculum contains Leishmania exosomes.

Figure 3

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L. infantum- and L. major-infected sand flies where fed in vitro through a chicken-skin membrane. Sand fly egests where collected and analyzed for exosomes by TEM and western blot. (A) Exosome vesicles isolated from L. infantum and L. major inocula. (B) Western blots of: vesicles isolated from L. infantum (Li) and L. major (Lm)-infected sand fly egests (4 experiments each) compared to L. infantum and L. major egested parasites (LiP and LmP), in vitro-isolated L. major exosomes (LmE) and uninfected sand fly inocula (Un). The results are representative of 8 independent experiments (4 experiments for each Leishmania species). See also Figure S7.

To confirm the identity of these vesicles, we first performed MS analyses. Searching Leishmania databases, we were able to detect 22 to 24 Leishmania-specific proteins and exosome markers including GP63, HSP83, HSP70, elongation factor 1-alpha and calmodulin (table S5). Searching the L. longipalpis database, only 3 to 5 proteins were detected, indicating that the amount of vesicles derived from the sand fly was negligible (table S5). Lastly, we performed GP63 western blots with vesicles isolated from several biological replicas of L. infantum and L. major sand fly inocula, compared to uninfected inocula and to parasites collected from the inocula. Different amounts of GP63 were detected among the replicas, indicating that the amount of exosomes injected by the sand fly into the bite site can vary (Fig. 3B).

Taken together, these results clearly demonstrate that exosomes secreted by either L. infantum or L. major promastigotes within the sand fly midgut are egested alongside parasites and delivered into the bite site. Our next question was how and to which extent Leishmania exosomes may influence the development of experimental cutaneous leishmaniasis.

Leishmania exosomes significantly enhances lesion development by favoring parasite replication

To understand the impact of Leishmania exosomes in the infection site, we co-injected mice footpads with 105 L. major metacyclic forms (META) along with 1 µg of midgut-isolated (Gut) or in vitro-isolated (Exo) L. major exosomes. We observed a significant and sustained exacerbation of the lesions up to 6 weeks post-infection (Fig. 4A). Taking the advantage of the similarity between L. major in vivo and in vitro-isolated exosomes, we used in vitro preparations which yields high amounts of exosomes to further explore our hypothesis. We co-injected mice footpads with L. major and in vitro-purified L. major exosomes, and monitored the lesions for up to 10 weeks, depending on the experiment. Mice injected with 5 × 106 stationary promastigotes (STAT) along with 10 µg of exosomes displayed significantly enhanced footpad swelling starting from week 2, which was sustained throughout the follow-up period (Fig. 4B). To better reproduce the sand fly bite, the experiment was repeated with culture-purified metacyclic promastigotes. Likewise, mice injected with 105 metacyclics (META) along with exosomes had a sustained and significant increase in their footpad swelling, starting from week 3 (Fig. 4B). These results indicate that exosomes may play a role in the biological process leading to lesion development caused by Leishmania.

Figure 4. Injection of Leishmania alongside in vivo- or in vitro-isolated exosomes significantly enhances mice footpad lesions.

Figure 4

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BALB/c mice were infected with 105 L. major metacyclics (META) or with 5 × 106 L. major stationary promastigotes (STAT) with or without 1 µg of midgut-isolated L. major exosomes (Gut) or 1 µg (A) and 10 µg (B) of in vitro-isolated L. major exosomes (Exo). (A, B) Lesions were monitored up to 10 weeks post-infection. (C–E) STAT and META groups from (B) on the 6th and 10th week post-infection, respectively. (C) Footpad images. (D) Lesion volumes. (E) Footpad parasite loads. Each data point represents the average + SEM of 6 to 8 mice per group and statistical significance at + p≤0.05, * p≤0.01, ** p≤0.001, *** p≤0.0001, ns, non-significant. FP, footpad. The results are representative of 1(A) or 2 (B–E) independent experiments.

At the end of both experiments, footpads were photographed (Fig. 4C) and their volumes calculated. Groups co-inoculated with Leishmania and exosomes exhibited a 3- to 4-fold increase in lesion volume compared to groups inoculated with parasites alone (Fig. 4D). In addition, the number of parasites recovered from footpads injected with metacyclics alongside exosomes was enhanced by more than 3-fold compared to the group injected with metacyclics alone (Fig. 4E). Of note, no significant differences in the footpad parasite loads were observed between STAT groups (Fig. 4E) - potentially due to the development of highly necrotic lesions as illustrated in Figure 3C. Indeed, in following experiments, the footpad parasitic load of non-necrotic lesions was found to be significantly higher in mice infected with STAT plus exosomes.

Disruption of exosome integrity diminishes their ability to exacerbate leishmaniasis

To investigate whether exosome integrity is important for the enhancing effect on lesion development, pelleted membranes from disrupted (D) or disrupted and boiled (D/B) exosomes (Fig. 5A and 5B) were injected in mice along with L. major STAT. Disruption and disruption with boiling caused a partial and total abrogation, respectively, of the enhancing effect of exosomes on lesion size (Fig. 5C) as well as a significant decrease in parasite loads compared to mice co-injected with intact exosomes (Fig. 5D). This indicates that exosome integrity is important for optimal delivery of parasite inflammatory factors. Interestingly, mice co-injected with D and D/B exosomes developed more ulcerative lesions at the end of 7 weeks compared to mice co-injected with intact exosomes (Fig. 5E). This effect may be due to exosome membranes that showed detectable levels of gp63 and potentially other molecules that could trigger inflammation directly (Fig. 5B). As expected, mice injected with exosomes alone did not develop lesions (Fig. 5C).

Figure 5. Exosome integrity is important for the enhancing effect on lesion development.

Figure 5

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BALB/c mice were infected with 5 × 106 L. major stationary promastigotes (STAT) with or without 10 µg of in vitro-isolated L. major exosomes, sonication-disrupted exosomes (D), disrupted and boiled exosomes (D/B) or with exosomes alone (Exo). Exosome preparations were prepared for TEM or submitted to SDS-PAGE before (total) and after ultracentrifugation (pellet). Their protein content was compared to whole parasite lysates (Pro). (A) Intact and disrupted exosome structures. (B) Top, total protein silver staining. Bottom, HSP83 and GP63 western blots. (C) Lesions were monitored up to 7 weeks post-infection. (B) Footpad parasite loads at week 7. (C) Footpad images at week 7. Each data point represents the average + SEM of 6 to 8 mice per group and statistical significance at + p≤0.05, * p≤0.01. FP, footpad. The results are representative of 2 independent experiments.

Leishmania exosomes exacerbate lesion development through the induction of inflammatory cytokines

To investigate whether the exacerbation effect of exosomes on lesion size and parasite replication was dose-dependent, we repeated the mice co-injection experiment using STAT and lower doses of in vitro-purified exosomes. Interestingly, we did not observe a strongly pronounced dose-response effect when we compared the concentrations of 1 µg and 5 µg to 10 µg, possibly due to the effectiveness of these vesicles even at lower concentrations (Fig. S7B). A stronger dose-dependent effect was achieved when we used the concentration of 0.1 µg (Fig. 6A and 6B). Since co-injection of 1 µg of exosomes with parasites is likely to be more physiological (this is twice the approximate amount of exosomes secreted by 5×106 parasites in vitro when incubated for 4 hours at 37°C), and generated enhanced but non-necrotic lesions up to 7 weeks post-infection (Fig. 6C, S7B and S7C), we decided to analyze immunological events in this group compared to parasites alone.

Figure 6. Leishmania exosomes exacerbate disease outcome by enhancing cytokine expression.

Figure 6

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(A) BALB/c mice were infected with 5 × 106 L. major stationary promastigotes (STAT) with or without in vitro-isolated L. major exosomes (0.1, 1 or 10 µg) and lesions were monitored. (B) Footpad parasite loads at week 6. (C) Images of footpads co-injected or not with 1 µg of exosomes 5 weeks post-infection. (D) Cytokine mRNA levels in draining lymph nodes 5 weeks post-infection. Symbols on top of each bar represent p values compared to the STAT group. Each data point represents the average + SEM of 4 to 8 mice per group and statistical significance at + p≤0.05, * p≤0.01, ** p≤0.001, *** p≤0.0001. FP, footpads, NI, non-infected, L.m., Leishmania major. The results are representative of 2 independent experiments. See also Figure S7.

We recovered the lymph nodes draining lesion sites at week 5 post-infection and evaluated their cytokine mRNA levels. The targeted cytokines were IFN-γ and IL-4, associated to Th1 and Th2 immune responses against Leishmania infection, respectively (Heinzel et al., 1991; Sacks and Noben-Trauth, 2002), IL-10, previously described to be produced by mice splenocytes after Leishmania exosome vaccination (Silverman et al., 2010b) and IL-17a, a hallmark of Th17 inflammation related to neutrophil infiltration in human and murine lesions (Boaventura et al., 2010; Lopez Kostka et al., 2009). In addition, we measured mRNA levels of IL-23, which is shown to be produced by Leishmania-infected dendritic cells (Lopez Kostka et al., 2009) and it is involved in the induction of IL-10 and IL-17 by human naïve T cells (Vanden Eijnden et al., 2005). Levels of IL-2 were also examined. All six cytokines were significantly enhanced in the group co-injected with exosomes in relation to the STAT group (Fig. 6D). Remarkably, IL-17a displayed the highest expression among the tested cytokines, suggesting it is highly induced by exosomes. In summary, the lymph node cytokine profile of mice co-injected with L. major and exosomes was characterized by enhanced inflammation, possibly mediated by Th17 cells.

DISCUSSION

Leishmania has evolved effective strategies to persistently infect the mammalian host (Olivier et al., 2012). In the past few years, our group and others have proposed exosome secretion as one of the strategies used by the parasite to orchestrate beneficial changes in the host environment ensuring a successful infection. Leishmania exosomes were shown to deliver virulence factors to host macrophages and to modulate the immune system creating an environment permissive for early infection (Hassani et al., 2011; Silverman et al., 2010a; Silverman and Reiner, 2011b). It is interesting to note that Leishmania species are early divergent eukaryotes that have retained the exosome secretory pathway through evolution, similarly to their mammalian counterparts. In vitro-isolated Leishmania exosomes are fairly similar in structure and content to exosomes derived from higher eukaryotes (Silverman et al., 2010a), which might be advantageous in their interaction with the host.

In this study, we demonstrated the secretion of exosomes by L. infantum and L. major in vivo, within the sand fly midgut. These exosomes were found to be authentic with respect to previously characterized in vitro-isolated Leishmania exosomes, and appeared to mirror the physiological state of the parasites from which they were derived. Although there are some differences, exosomes derived from both species shared a number of pro-inflammatory molecules, which might be responsible for their effect on lesion development. Promastigotes from both species residing in the sand fly midgut, including the anterior midgut, appeared to secrete exosomes via three different routes. First, from MVBs, which suggested that the mechanism of Leishmania exosome biogenesis is reasonably similar to the one of mammalian cells. Second, from flagellar pockets, which are organelles involved in protein endocytosis and exocytosis of kinetoplastids (Field and Carrington, 2009). Third, we frequently observed vesicles emerging from discontinued membrane surfaces, despite the microtubular network surrounding the cell body of Leishmania. Interestingly, we observed MVBs fusing directly with the flagellar pocket membrane, which might be another way of exosome release into the outer milieu. Although vesicles secreted by different routes showed a similar structure, observed by TEM, it remains a question whether their contents diverge.

Previous works revealed that metacyclic promastigotes are regurgitated from the sand fly anterior midgut into the injection site during blood feeding (Schlein et al., 1992; Volf et al., 2004; Warburg, 2008). In addition, it has been shown that sand fly salivary proteins and the promastigote secretory gel are co-deposited into the bite site, exacerbating Leishmania infection (Gomes and Oliveira, 2012; Rogers et al., 2009; Rogers et al., 2004). We hypothesized that Leishmania exosomes may also be egested alongside parasites. Using a system where infected sand flies were fed through a chicken membrane, we were able to collect and study the egested material for the presence of exosomes. Our results clearly demonstrated that these vesicles are part of the sand fly inocula, hence being delivered alongside the parasite during the sand fly bite. In addition, our results place exosomes in a natural context and show their ability to reach the host and influence the early events in the establishment of the infection.

The addition of intact exosomes to L. major infections exacerbated the lesions significantly, as well as facilitated parasite replication. Recently, using an in vivo air-pouch model, we reported that exosomes are able to induce an inflammatory recruitment of neutrophils and macrophages to the injection site (Hassani et al., 2014). The exacerbated pathology in footpad infections containing exosomes supports our results and suggests that exosomes may induce inflammatory events that favor parasite replication.

To begin to understand the immunological events triggered by exosomes when co-injected with Leishmania, we collected draining lymph nodes at week 5 post-infection and measured their cytokine mRNA levels. Mice co-injected with L. major exosomes expressed significantly increased levels or IL-4 and IFN-γ, corroborating the exacerbation of lesion size as well as increased parasite replication. In a previous work, BALB/c mice vaccinated with L. major exosomes prior to challenge with L. major showed higher frequencies of IL-4-producing CD4+ T cells in both spleen and draining lymph nodes and disease exacerbation (Silverman et al., 2010b). In our experiments, we observed an overinduction of IL-17a over IL-4, which can be explained by the fact that we co-injected exosomes with parasites instead of using them for vaccination. IL-17a has been involved in Leishmania infection and it is a hallmark of Th17 inflammation related to neutrophil infiltration in human and murine lesions (Boaventura et al., 2010; Lopez Kostka et al., 2009). These results are in accordance with previous data showing that IL-17a is produced by susceptible mice infected with L. major, despite Th2 polarization (Lopez Kostka et al., 2009). Regarding IL-10, it has been shown that this cytokine is highly produced by splenocytes from C57BL/6 mice vaccinated with in vitro-isolated L. donovani exosomes prior to challenge with L. donovani (Silverman et al., 2010b). In our experiments, we also observed overinduction of IL-10 when we added exosomes. Of note, it is well established that both IL-4 and IL-10 are associated to susceptibility to Leishmania infections (Sacks and Anderson, 2004).

Our study established the in vivo secretion of Leishmania exosomes and placed them in the parasite’s natural life cycle revealing new features of Leishmania pathogenesis never addressed before. We demonstrated that midgut-secreted exosomes are part of the sand fly inocula and can regulate the immune system and exacerbate disease outcome when co-inoculated alongside parasites, adding to the repertoire of virulence factors involved in vector-transmitted infections and bringing us a step closer to fully elucidating the observed potency of these infections.

EXPERIMENTAL PROCEDURES

Ethics Statement

Animal experiments were performed in accordance with the Canadian Council on Animal Care (CCAC) Guidelines, approved by the Institutional Animal Care and Use Committees at the McGill University under ethics protocol number 4859.

Parasites

A recent isolate of Leishmania infantum (MCAN/BR/09/52) obtained from a dog with advanced canine leishmaniasis from Natal, Brazil, and the wild type L. major strain NIH S (MHOM/SN/74/Seidman) clone A2 (Joshi et al., 2002), were used for sand fly infections. Procyclic promastigotes were cultured at 26°C in Schneider’s Drosophila Medium (Gibco-BRL, Grand Island, NY) supplemented with 20% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µl/ml streptomycin (complete Schneider’s), for 2 passages before the infections. For mice infections and in vitro exosome purifications, the wild type L. major strain NIH S (MHOM/SN/74/Seidman) clone A2 (Joshi et al., 2002) was used. Promastigotes were cultured in complete Schneider’s Medium at 26°C and stationary phase cultures containing infective forms were used to infect female BALB/c mice 6–8 weeks old. For the purification of metacyclic promastigotes, L. major stationary promastigotes were incubated for 15 minutes at room temperature with 50 µg/ml of peanut agglutinin (Sigma Aldrich, St. Louis, MO). The sample was then centrifuged at low speed to precipitate procyclic promastigote clumps and the supernatant containing metacyclic promastigotes was recovered.

Mice Infections

All experiments with mice were carried out in pathogen-free housing and in accordance to the regulations of the Canadian Council of Animal Care, approved by the McGill University Animal Care Committee. Female BALB/c mice (6 to 8 weeks old) were infected in the right hind footpad with 5×106 stationary phase L. major promastigotes or with 105 purified metacyclic promastigotes with or without 1, 5 or 10 µg of in vitro purified exosomes, depending on the experiment. Disease progression was monitored by measuring footpad swelling weekly with a metric caliper, up to 10 weeks post-infection. At the end of each experiment, footpads were imaged for determination of lesion areas (Photoshop CS6) and volumes (multiplying lesion area by lesion thickness), and processed for determination of the parasite burden.

Sand fly infections and preparation of midgut samples

Colony-bred 2 to 4 day-old Lutzomyia longipalpis females were infected by artificial feeding on defibrinated rabbit blood (Spring Valley Labs, Sykesville, MD) containing 5×106 procyclic promastigotes per ml of blood, for 3 h in the dark as described elsewhere (Kamhawi et al., 2000). Fully blood-fed flies were separated and maintained at 26°C with 75% humidity and were provided 30% sucrose. After 10–12 days, sand flies with mature infections containing active metacyclic promastigotes were anesthetized with CO2, washed in 5% soap solution and rinsed in PBS prior to dissections. Uninfected sand flies were used as controls. Midguts were dissected and fixed overnight with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for TEM, or opened longitudinally in a drop of PBS to recover their luminal content for vesicle isolation (midgut lavages).

Transmission Electron Microscopy (TEM)

Vesicles purified from midgut lavages or from L. major axenic cultures were coated directly on formvar carbon grids, fixed with 1% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 minute and stained with 1% uranyl acetate for 1 minute. For ultrastructure analysis, dissected midguts from infected or uninfected sand flies were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer overnight. After washes in the same buffer, samples were post-fixed with osmium tetroxide and dehydrated in a graded acetone series prior to the embedding in epoxy resin. Ultrathin sections (70–80 nm) were cut from resin blocks using a Reichert-Jung Ultracut E Ultramicrotome (Biel, Switzerland). Formvar grids covered with isolated vesicles or with ultrathin sections were visualized in the FEI Tecnai 12 120 kV transmission electron microscope. Images were taken with the AMT XR-80C CCD Camera System (Facility for Electron Microscopy Research, McGill University).

Isolation of vesicles from sand fly midgut lavages

Midgut lavages were initially centrifuged at 3,000 RPM for separation of parasites and cell debris, followed by a centrifugation at 13,000 RPM for separation of bacteria. Due to the small volume of the sand fly midgut lavages, vesicles were purified with the ExoQuick exosome precipitation reagent (Mountain View, CA), following the manufacturers’ protocol. ExoQuick is a one-step, polymer-based method for precipitation of vesicles of 60–180 nm in diameter from fluids. After ExoQuick purifications, an average of 10 µg of vesicles was obtained from infected lavages per experiment, which was measured with the MicroBCA protein assay kit (Thermo scientific, Rockford, IL). Uninfected lavages yielded lower concentrations as expected.

Liquid Chromatography-Mass Spectrometry (LC-MS/MS)

LC-MS/MS was performed at the Institute de Recherches Cliniques de Montréal (IRCM, University of Montreal, Canada). Proteins derived from midgut-isolated materials were precipitated with 15% trichloroacetic acid (TCA)/acetone and processed for LC-MS/MS analysis. After precipitation, in solution digestion was performed by the addition of trypsin at a ratio of 1:25 protease: protein. After an overnight incubation at 37°C, the reactions were quenched by the addition of formic acid to a final concentration of 0.2% and cleaned with C18 Zip Tip pipette tips (Millipore, Billerica, MA), before mass spectrometry analysis. Extracted peptides were injected onto a Zorbax Extended-C18 desalting column (Agilent, Santa Clara, CA) and subsequently chromatographically separated on a Biobasic 18 Integrafrit capillary column (Thermo Scientific, Rockford, IL) on a Nano High-Performance liquid chromatography system (1100 series unit; Agilent, Santa Clara, CA). Eluted peptides were electrosprayed as they exited the capillary column and were analyzed on a QTRAP 4000 linear ion trap mass spectrometer (SCIEX/ABI, Framingham, MA).

Protein database search

Individual sample tandem mass spectrometry spectra were peak listed using the Distiller version 2.1.0.0 software (www.matrixscience.com/distiller) with peak picking parameters set at 1 for signal noise ratio and at 0.3 for correlation threshold. The peak-listed data was then searched against the NCBI database with the Mascot software version 2.3.02 (Matrix Science, London, UK). Mascot was set up to search the L. infantum database (txid5671, 16,895 proteins) or the L. longipalpis database (txid7200, 1,138 proteins) with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 1.5 Da. Carbamidomethyl was specified in both search engines as a fixed modification. Oxidation of methionine residues was specified in Mascot as a variable modification. Scaffold software version 4.0.6.1 (Proteome Software Inc., Portland, OR) was used to validate MS/MS peptide and protein identifications. Identification of peptides was accepted if it could be established at greater than 95.0% probability. Identification of proteins was accepted if it could be established at greater than 95.0% probability and contained at least 2 identified peptides. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. The final number of peptides per protein was represented by the average of the biological replicas after normalization to the total number of peptides.

Bioinformatic analyses

Gene ontology (GO) annotations were attributed using Blast2GO (Gotz et al., 2008). The initial Blastp step was performed against NCBI nonredundant database and high scoring top blast hits were retrieved and used for annotation with Blast2GO default parameters. Signal peptide predictions were performed using SignalP 4.1 (Petersen et al., 2011). Prediction of unconventionally secreted proteins was performed using SecretomeP 2.0 server (Bendtsen et al., 2004). L. infantum interacting exosome proteins were mapped based on the predicted interactome of Leishmania infantum (Rezende et al., 2012). Interaction network layout was created using Cytoscape 2.8.3 (Smoot et al., 2011).

Egestion experiments

To obtain the egests of L. infantum- and L. major-infected sand flies, groups of 10–12 day infected flies were fed through a chicken skin system as reported elsewhere (Rogers et al., 2004) on RPMI or rabbit plasma (Spring Valley Labs, Sykesville, MD) used after a high-speed spin at 100,000 g for 1 hour to remove vesicles. Egests were initially centrifuged at 3,000 RPM for separation of parasites and cell debris, followed by a centrifugation at 13,000 RPM for separation of bacteria. Vesicles were purified with the ExoQuick exosome precipitation reagent (Mountain View, CA), following the manufacturers’ protocol. After ExoQuick purifications, concentration was measured with the MicroBCA protein assay kit (Thermo scientific, Rockford, IL).

Purification of exosomes from Leishmania in vitro

Purification of Leishmania exosomes was performed according to standard methods for mammalian cells (Thery et al., 2006). L. major stationary phase parasites were washed twice with PBS, resuspended in RPMI 1640 medium (Life Technologies, Rockville, MD) without FBS and phenol red at a concentration of 108 promastigotes/ml, and incubated for 4 hours at 37°C for the release of exosomes in the culture medium. It has been described that a temperature shift significantly increases the release of exosomes by Leishmania in vitro (Hassani et al., 2011; Silverman et al., 2010a). We used this method to improve our yields. Parasite viability was measured by propidium iodide staining before and after the incubation at 37°C. At the end of a 4-hour incubation, the sample was centrifuged at 3,000 RPM to clear out parasites, at 10,000 RPM to clear out debris, and filtered through 0.45 µm followed by 0.20 µm syringe filters. Next, exosomes were pelleted by a 1-hour centrifugation at 100,000 g and resuspended in exosome buffer (137 mM NaCl, 20 mM Hepes, pH 7.5). For further purification, exosomes were layered in a linear sucrose gradient (0 to 2 M sucrose) and centrifuged at 65,000 RPM for 1.5 hour. Ten fractions of 1 ml were collected and exosomes or their protein contents were detected by TEM or Western blot in fractions 4, 5 and 6, corresponding to the concentrations of 0.8 to 1.2 M of sucrose and densities of 1.10 to 1.15 g/ml. Exosome-containing fractions were combined, pelleted, resuspended in endotoxin-free PBS and dosed for inoculation of mice. For disruption, exosome preparations were sonicated (6 cycles of 25 seconds at 25-second intervals on ice), or sonicated and boiled for 5 minutes.

Determination of footpad parasite loads by the limiting dilution assay

Footpads were surface-sterilized with a chlorine dioxide based disinfectant followed by ethanol 70% for 5 minutes. After several washes in PBS, footpads were sliced, transferred to a glass tissue homogenizer containing 6 ml of PBS, and manually homogenized until complete tissue disruption was achieved. The final homogenate was then centrifuged at 3,000 × g for 5 minutes, resuspended in 10 ml of PBS and titrated by the Bradford Protein Assay. Homogenate volumes were adjusted (and further diluted when necessary) and 100 µl were added in duplicates to 96-well plates containing 100 µl of complete Schneider’s medium in each well (twenty-four 2-fold dilutions for each duplicate). Plates were kept at 28°C until examined after 7–10 days, when the highest dilutions at which promastigotes were observed were recorded. Parasite loads were expressed as number of parasites per footpad.

Western blots

Midgut materials, parasites or exosome preparations were dosed with the Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, IL) or with the Bradford Protein Assay Kit (Bio-Rad, Mississauga, ON, Canada) and resuspended directly in SDS sample buffer containing bromophenol blue and beta-mercapto-ethanol. Proteins were subjected to SDS-PAGE and transferred to PVDF membranes (Perkin Elmer, Waltham, MA). After a 1-hour blocking in TBS-0.05% Tween 20 (TBST) containing 5% fat-free milk, membranes were incubated with the following specific antibodies against: HSP83 (Greg Matlashewski, McGill University, Canada), GP63 (Robert W. McMaster, University of British Columbia, Vancouver, Canada) and HGPRT (Armando Jardim, McGill University, Canada). Proteins were detected with specific IgG horseradish peroxidase-conjugated antibodies (GE Healthcare, Baie d’Urfe, QC, Canada) and subsequently visualized by the ECL Western Blot Detection System (GE Healthcare).

Quantitative RT-PCR (qRT-PCR)

Total RNA from mice footpads or lymph nodes was extracted with TRIzol reagent (Life Technologies), treated with RQ1 DNase (Promega) for clearance of DNA contaminants and purified using the RNeasy Mini Kit (Qiagen). 2–5 µg of DNA-free RNA was reverse transcribed using Superscript III Reverse Transcriptase and random hexamers (Invitrogen). Standardized amounts of cDNA and custom designed primers were mixed with SYBR Green Supermix (Bio-Rad) and the PCRs were performed in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) according to manufacturer’s protocol. Results were analyzed by the ΔΔCt method.

Supplementary Material

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Highlights.

  • *

    Leishmania secretes exosomes within the sand fly midgut

  • *

    Leishmania and its exosomes are co-egested during the insect’s blood meal

  • *

    Leishmania exosomes exacerbate cutaneous leishmaniasis pathology

  • *

    Leishmania exosomes overinduce inflammatory cytokines, in particular IL-17a

ACKNOWLEDGEMENTS

This research was supported by grants from the Canadian Institutes of Health Research (CIHR) to M.O and by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, NIH, to S.K. M.O. is a Burroughs Welcome Fund Awardee in Molecular Parasitology. V.D.A. was supported by the Dr. David T. W. Lin Postdoctoral Fellowship, McGill University Faculty of Medicine, and by the Research Institute of the McGill University Health Centre. M.O. and V.D.A. are members of the Québec province CHPI-FQRNT network in Host-Parasite Interaction. We thank the Facility for Electron Microscopy Research, McGill University, for assistance with TEM and the Institut de Recherches Cliniques de Montréal for the proteomics. We thank Dr. Jesus Valenzuela (NIAID, NIH) for his support; Mr. Claudio Meneses (NIAID, NIH) for providing the sand flies and Dr. Greg Matlashewski (McGill University), Dr. Robert W. McMaster (University of British Columbia) and Dr. Armando Jardim (McGill University) for kindly providing us with antibodies.

Footnotes

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AUTHOR CONTRIBUTIONS

V.D.A., H.A.S., S.T., K.H., S.K and M.O. designed and carried out the experiments. V.D.A., H.A.S., K.H., S.T., S.K. and M.O. analyzed the data. V.D.A, S.K. and M.O. wrote the paper. All authors discussed the results and commented on the manuscript.

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