Leishmania Exosomes/Extracellular Vesicles Containing GP63 Are Essential for Enhance Cutaneous Leishmaniasis Development Upon Co-Inoculation of Leishmania amazonensis and Its Exosomes.

Alonso Da Silva Lira Filho,Martin Olivier,Pauline Clément,Kwang Poo Chang,Emanuella Francisco Fajardo

doi:10.3389/fcimb.2021.709258

Abstract

Protozoan parasites of the genus Leishmania are transmitted by the bite of infected sand flies leading to a wide range of diseases called leishmaniasis. Recently, we demonstrated that Leishmania spp.-derived exosomes/extracellular vesicles (EVs/LeishEXO) were released in the lumen of the sand fly midgut and to be co-egested with the parasite during the blood meal and that LeishEXO were found to stimulate an inflammatory response conducting to an exacerbated cutaneous leishmaniasis, also it was shown that these vesicles cargo important virulence factors like GP63. Thus, this study aimed to confirm through morphological and proteomic analysis a novel model specificity utilizing another set of GP63-altered Leishmania amazonensis parasite strains. Consequently, we proposed to further study the impact of different GP63 vesicle expression levels on their ability to modulate innate inflammatory cell responses, and finally to determine the importance of GP63 vesicle content on the exacerbation of the cutaneous Leishmania spp. pathology after their host co-inoculation. Our results revealed that the protein composition of extracted extracellular vesicles were similar to each other and that GP63 was the sole virulence factor changed in the exosomes composition confirming the specificity of the chosen novel model. We further demonstrated that vesicles with different GP63 EVs cargo displayed distinctive macrophage immunomodulatory capabilities at both gene and protein expression in vitro. Finally, we showed their diverse impact on the Leishmania spp. cutaneous pathology in an in vivo setting and confirmed GP63 as a primordial component of the ability of these EVs in augmenting the inflammatory cutaneous response in Leishmania spp. infection. Our findings provide new insight on the immune response happening in cutaneous leishmaniasis, shade light on the mechanism behind the host-pathogen interaction occurring in the initial moments of infection, thus creating the opportunity of using them as the target of new pharmacological treatments and vaccinations.

Highlights

Leishmania spp. is a trypanosomatid protozoan parasite transmitted by the bite of infected female phlebotomine sandflies that can lead to a variety of different diseases called Leishmaniasis
In order to assess the importance of GP63 on the immune modulation of the cutaneous leishmaniasis, a set of transgenic parasites of the species L. amazonensis (LV78, MPRO/ BR/72/M1845) had their endogenous GP63 levels up-or downregulated by episomal expression of sense and antisense GP63 RNA was chosen for this study (Chen et al, 2000)
GP63 is the main subject of many studies that identified it as a key virulence factor capable of many diverse effects on the macrophage cell biology and its response to the parasite infection (Brittingham et al, 1995; Yao et al, 2003; Contreras et al, 2010; Olivier et al, 2012)

Summary

Introduction

Leishmania spp. is a trypanosomatid protozoan parasite transmitted by the bite of infected female phlebotomine sandflies that can lead to a variety of different diseases called Leishmaniasis. From a self-healing cutaneous lesion (Cutaneous Leishmaniasis) to its possibly lethal systemic visceral form (Visceral Leishmaniasis), it is considered by the World Health Organization a neglected vector-borne tropical infection.. Exosomes are endosomal vesicles of 30-150 nm of diameter produced by the majority of eukaryotic and prokaryotic cells (Johnstone et al, 1987). These extracellular vesicles have been described to have a role in many physio/pathological contexts like cell-cell communication (Johnstone et al, 1987), immune (Turpin et al, 2015), kidney (Ferná ndez-Llama et al, 2010), cancer (Skog et al, 2008), and infectious diseases (Hosseini et al, 2013). Exosomes are produced by Leishmania spp. and are considered part of its secretome containing the previously mentioned GP63, and lipophosphoglycan (LPG), and elongation factor 1 (EF-1) (Hassani et al, 2011; Olivier et al, 2012; World Health Organization, 2014)

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