Proteomic analysis of exosomes derived from procyclic and metacyclic-like cultured Leishmania infantum chagasi

Leishmania infantum chagasi is the primary etiological agent of visceral leishmaniasis in Latin America, a lethal disease that afflicts hundreds of thousands of people worldwide every year. Previous studies have shown that the parasite releases microvesicles known as exosomes, which prolong and exacerbate infection in the vertebrate vector. However, little is known of their role in the insect vector, the sand fly Lutzomyia longipalpis. Exosomes were isolated from cultured L. i. chagasi in logarithmic (procyclic) (LOG) and stationary phase (metacyclic-like) (STAT) growth stages, which are the parasite stages found in the vector, and submitted to proteomic analysis. Our studies showed that exosomes from LOG and STAT L. i. chagasi display discrete protein profiles. The presence of approximately 50 known virulence factors was detected, including molecules for immunomodulation and evasion (GP63, EF1 alpha, Oligopeptidase), increased pathogenicity (Casein kinase, KMP-11, Cysteine Peptidase and BiP) and parasite protection (Peroxidoxin). Additionally, the majority of ontological terms were associated with both exosome phases, and no substantial ontological enrichment was observed associated with any of the two exosomal stages. We demonstrated that LOG exosomes show a marked increase in protein number and abundance, including many virulence factors, compared to STAT L. i. chagasi exosomes. Significance: The knowledge of the role of Leishmania exosomes on leishmaniasis opened up a new world of potential and complexity regarding our understanding of the disease. In Brazil the majority of visceral leishmaniasis cases are caused by the parasite Leishmania infantum chagasi and transmitted by the vector Lutzomyia longipalpis. While Leishmania exosomes were found to play an active role in the mammalian host, little is understood about their effects on the sand fly, or how they might impact on the insect infection by the parasite. For this reason, we isolated exosomes from two developmental stages of L. i. chagasi that occur within the insect with a view to identifying and describing the alterations they undergo. We have identified many hundreds of proteins within both exosome phases and have developed a structure by which to examine potential candidates. Our findings regarding the composition of the exosome proteome raise many questions regarding their function and provide compelling evidence that exosomes play an active role in the parasite's development within the sand fly.