Endocytosis and Exocytosis in Leishmania amazonensis Are Modulated by Bromoenol Lactone

Abstract

In the protozoan pathogen Leishmania, endocytosis, and exocytosis occur mainly in the small area of the flagellar pocket membrane, which makes this parasite an interesting model of strikingly polarized internalization and secretion. Moreover, little is known about vesicle recognition and fusion mechanisms, which are essential for both endo/exocytosis in this parasite. In other cell types, vesicle fusion events require the activity of phospholipase A 2 (PLA 2 ), including Ca 2+ -independent iPLA 2 and soluble, Ca 2+ -dependent sPLA 2 . Here, we studied the role of bromoenol lactone (BEL) inhibition of endo/exocytosis in promastigotes of Leishmania amazonensis. PLA 2 activities were assayed in intact parasites, in whole conditioned media, and in soluble and extracellular vesicles (EVs) conditioned media fractions. BEL did not affect the viability of promastigotes, but reduced the differentiation into metacyclic forms. Intact parasites and EVs had BEL-sensitive iPLA 2 activity. BEL treatment reduced total EVs secretion, as evidenced by reduced total protein concentration, as well as its size distribution and vesicles in the flagellar pocket of treated parasites as observed by TEM. Membrane proteins, such as acid phosphatases and GP63, became concentrated in the cytoplasm, mainly in multivesicular tubules of the endocytic pathway. BEL also prevented the endocytosis of BSA, transferrin and ConA, with the accumulation of these markers in the flagellar pocket. These results suggested that the activity inhibited by BEL, which is one of the irreversible inhibitors of iPLA2, is required for both endocytosis and exocytosis in promastigotes of L. amazonensis.

Lipid profile of Trichinella papuae muscle-stage larvae

“…In case of PLA2, it is an important energy source enzyme and crucial in the development of Clonorchis sinensis mediated hepatic fibrosis 61 . PLA2 has been reported its role on membrane remodelling and vesicle secretion in several organisms for example P. falciparum 62 and Leishmania amazonensis 63 . Plasmodial PLA2 involves the egress of merozoites from liver-stage schizonts 64 and merozoite invasion of red blood cells 58 .…”

The Leishmania donovani LDBPK_220120.1 Gene Encodes for an Atypical Dual Specificity Lipid-Like Phosphatase Expressed in Promastigotes and Amastigotes; Substrate Specificity, Intracellular Localizations, and Putative Role(s)

“…Interestingly, in dividing and non-dividing morphological forms of promastigotes in culture, Ld TyrPIP_22 was localized at the periphery of the cell body ( Figure 5 , Figures S5Ac, d , S6 and S9 ) in areas rich in Ld Actin ( Figures 5A–D insets) and microtubules ( Figures S6 ) or stained with the FM4-64 dye ( Figure S7 ) ( Vince et al., 2008 ). Occasionally, Ld TyrPIP_22 epitopes were also detected in internal tubular/filament-like structures ( Figure 5D , bottom inset, Figures S5Ab , S7b ) which could be part of the tubular MTV endosome ( Halliday et al., 2019 ; Wang et al., 2020 ) and at the posterior region of the cell body ( Figure 5A , Figure S7b ), area where internalized/endocytosed cargo accumulates in late endosomes ( Ghedin et al., 2001 ; Fernandes et al., 2020 ). Lower intensity punctate staining was also observed along the entire flagellum in most parasites ( Figures 5B and S5Aa, c, d and S5Bb ).…”

“…These morphologies, following nomenclature used for description of the flagellated promastigote forms observed in the midgut of sandflies (Sunter and Gull, 2017), could be assigned as: 1) dividing procyclic-like with short cell body (6.5-11.5 mm) and flagellum shorter than the cell body; 2) leptomonad-like with cell body between 6.5-11.5 mm and longer flagellum; 3) nectomonad-like with cell body longer than 12 mm and long flagellum and 4) the metacyclic-like with short (8 mm) and slender cell body and long flagellum (>2X longer than the cell body). Interestingly, in dividing and non-dividing morphological forms of promastigotes in culture, LdTyrPIP_22 was localized at the periphery of the cell body (Figure 5, Figures S5Ac, d epitopes were also detected in internal tubular/filament-like structures (Figure 5D, bottom inset, Figures S5Ab, S7b) which could be part of the tubular MTV endosome (Halliday et al, 2019;Wang et al, 2020) and at the posterior region of the cell body (Figure 5A, Figure S7b), area where internalized/ endocytosed cargo accumulates in late endosomes (Ghedin et al, 2001;Fernandes et al, 2020). Lower intensity punctate staining was also observed along the entire flagellum in most parasites (Figures 5B and S5Aa,c,d and S5Bb).…”

Neglected Zoonotic Diseases: Advances in the Development of Cell-Penetrating and Antimicrobial Peptides against Leishmaniosis and Chagas

“…The detailed mechanism of AMPs and/or CPPs internalization is not fully clear, but some hints have been elucidated [13]. Parasite phospholipases (PLA2) are involved in regulating the vesicular traffic, by modifying the membrane phospholipids and inducing the membrane deformation to generate the vesicles [29]. Inhibiting the PLA2 activity with bromoenol lactone blocks the trafficking of proteins such as transferrin or albumin.…”