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

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.

Proteome and morphological analysis show unexpected differences between promastigotes of Leishmania amazonensis PH8 and LV79 strains

“…Interestingly, enolase was recently described in exosomes shed by L . amazonensis promastigotes [ 16 ]. It is possible that the increase in enolase observed in PH8 promastigotes contributes to the higher infectivity of this strain.…”

“…Besides, GP63 present in exosomes enhances cutaneous leishmaniasis in L . amazonensis experimental model [ 16 ]. In the extracellular matrix, it may cleave collagen and fibronectin, helping in promastigote movement [ 58 ].…”

“…LPG is essential for adhesion of some Leishmania species to some vectors [8], as well as for phagocytosis and survival inside the macrophage [12]. Another important membrane-anchored virulence factor is metalloproteinase GP63, a protein involved in phagocytosis, resistance to complement lysis and subversion of macrophage signaling and activation [13][14][15][16][17].…”

Leishmania parasites exchange drug-resistance genes through extracellular vesicles

“…Extracellular vesicles (EVs) are nanometric membraneenclosed particles released into the extracellular space by organisms belonging to almost all living kingdoms; they serve as mediators of intercellular communication (Dong et al, 2021;Ya ´n ˜ez- Mo ´et al, 2015). In this way, EVs have emerged as key players in Leishmania biology by leading to, inter alia, an enrichment of parasite populations with key virulence factors (i.e., GP63) during the first moments of infection, promotion of parasite survival, or the triggering of an exacerbation of the disease (Atayde et al, 2015;da Silva Lira Filho et al, 2022;Dey et al, 2018;Hassani et al, 2014;Silverman et al, 2010). Recently, our group made the discovery that small EVs contain specific protein signatures reflecting the drug-resistance profile of the parental Leishmania strain from which they originate (Douanne et al, 2020a).…”

Revisiting Leishmania GP63 host cell targets reveals a limited spectrum of substrates

“…The availability of a genetically-defined L. major GP63 defective mutant ( Δgp63 ) (13) and of transgenic GP63-altered L. amazonensis and L. mexicana (42, 43) was instrumental to unravel the role played by GP63 in the modulation of host phenotypes/responses associated to Leishmania infection (21, 30, 31, 33, 36, 38, 43-47). In addition, the availability of these tools allowed to further understand how Leishmania subverts and manipulates host cell processes to ensure its survival and replication, through the identification of several GP63 substrates (21, 30, 31, 33, 36, 38, 43-45).…”