An exosome-based secretion pathway is responsible for protein export fromLeishmaniaand communication with macrophages

Abstract

SummarySpecialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37°C ± pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation.

Proteomic analysis reveals different composition of extracellular vesicles released by two Trypanosoma cruzi strains associated with their distinct interaction with host cells

“…It has been reported that cruzipain activity remains in the supernatant of parasite culture medium after high-speed centrifugation at 100,000 × g [57]. Secretome analyses of other trypanosomatids [13,14,38] such as Trypanosoma brucei, Leishmania donovani, and Leishmania major have shown that a large proportion, if not all of the secreted proteins are released as membrane-bound vesicles [58–60], similar to mammalian exosomes. Interestingly, in L. donovani, the composition and number of secreted vesicles seems to be regulated by conditions mimicking infection of host cells [60].…”

Sir2-Related Protein 1 fromLeishmania amazonensisis a glycosylated NAD+-dependent deacetylase

“…We provide evidence that LaSir2RP1 is secreted from both developmental stages of L. amazonensis. Thus, it would be interesting to determine whether LaSir2RP1 is secreted in a soluble form, either inside of or anchored to the membrane of vesicles/exosomes (Silverman et al 2010), since it does not present the classical XDXL motif at the C-terminus, as characteristic for ER/ Golgi resident proteins from protozoa to mammals (Munro and Pelham, 1987;Ilgoutz et al 1999), but was found dispersed in the parasite within vesicle-like structures. Accordingly, the secretory apparatus in parasitic kinetoplastids is organized in a similar way to those in mammalian cells and sequencing of the genes encoding some proteins of the kinetoplastid secretory pathway revealed that the presence of an N-terminal anchor signal is an important feature shared by these proteins (Sakaguchi et al 1992;McConville et al 2002).…”

The languages of parasite communication

“…The definition of excreted molecules was rather vague, essentially encompassing all molecules that did not appear to follow classical secretion pathways. In the past few years, however, several parasites have been shown to produce membrane-bound vesicles that increase their chances of survival and transmission [10][11][12][29][30][31][32][33][34]. In theory, there are many ways in which the contents of these vesicles can be delivered to cells within the host.…”

“…One is the ability to recruit macrophages to the inoculation site; the other is to stimulate the conversion of arginine to polyamines, which are required for growth of Leishmania, rather than nitric oxide, which activates macrophages and promotes parasite killing [49]. Leishmania promastigotes were among the first examples of parasites that were shown to extrude exosomes in culture [31][32][33]. In common with extracellular vesicles from Trichomonas and T. cruzi, these are enriched for specific fragments of tRNAs and rRNAs [33].…”

“…Very recently, it was demonstrated that exosomes with the same composition as culture-derived exosomes are produced by parasites in the sand fly and that these enhance the parasite load when co-injected with parasites [34]. In common with the other organisms described above, exosomes from Leishmania manipulate cytokine secretion, in this case enhancing the production of pro-inflammatory cytokines IL8 and IL17a, while inhibiting the production of TNFα, IL10 and IL12p70 [31,32]. It has also been reported that infection with L. donovani lowers the expression of a microRNA, miR-122, in the liver, resulting in lowered serum cholesterol [50].…”