title: Cooperation between apoptotic and viable metacyclics enhances the pathogenesis of Leishmaniasis.
abstract: Mimicking mammalian apoptotic cells by exposing phosphatidylserine (PS) is a strategy used by virus and parasitic protozoa to escape host protective inflammatory responses. With Leishmania amazonensis (La), apoptotic mimicry is a prerogative of the intramacrophagic amastigote form of the parasite and is modulated by the host. Now we show that differently from what happens with amastigotes, promastigotes exposing PS are non-viable, non-infective cells, undergoing apoptotic death. As part of the normal metacyclogenic process occurring in axenic cultures and in the gut of sand fly vectors, a sub-population of metacyclic promastigotes exposes PS. Apoptotic death of the purified PS-positive (PS(POS)) sub-population was confirmed by TUNEL staining and DNA laddering. Transmission electron microscopy revealed morphological alterations in PS(POS) metacyclics such as DNA condensation, cytoplasm degradation and mitochondrion and kinetoplast destruction, both in in vitro cultures and in sand fly guts. TUNEL(POS) promastigotes were detected only in the anterior midgut to foregut boundary of infected sand flies. Interestingly, caspase inhibitors modulated parasite death and PS exposure, when added to parasite cultures in a specific time window. Efficient in vitro macrophage infections and in vivo lesions only occur when PS(POS) and PS-negative (PS(NEG)) parasites were simultaneously added to the cell culture or inoculated in the mammalian host. The viable PS(NEG) promastigote was the infective form, as shown by following the fate of fluorescently labeled parasites, while the PS(POS) apoptotic sub-population inhibited host macrophage inflammatory response. PS exposure and macrophage inhibition by a subpopulation of promastigotes is a different mechanism than the one previously described with amastigotes, where the entire population exposes PS. Both mechanisms co-exist and play a role in the transmission and development of the disease in case of infection by La. Since both processes confer selective advantages to the infective microorganism they justify the occurrence of apoptotic features in a unicellular pathogen.
authors.affiliation: Experimental Medicine Division, National Cancer Institute, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: João Luiz Mendes
authors.initials: JL
authors.lastname: Wanderley
authors.forename: Lucia Helena
authors.initials: LH
authors.lastname: Pinto  da Silva
authors.forename: Poliana
authors.initials: P
authors.lastname: Deolindo
authors.forename: Lynn
authors.initials: L
authors.lastname: Soong
authors.forename: Valéria Matos
authors.initials: VM
authors.lastname: Borges
authors.forename: Deboraci Brito
authors.initials: DB
authors.lastname: Prates
authors.forename: Ana Paula Almeida
authors.initials: AP
authors.lastname: de Souza
authors.forename: Aldina
authors.initials: A
authors.lastname: Barral
authors.forename: José Mario de Freitas
authors.initials: JM
authors.lastname: Balanco
authors.forename: Michelle Tanny Cunha
authors.initials: MT
authors.lastname: do Nascimento
authors.forename: Elvira Maria
authors.initials: EM
authors.lastname: Saraiva
authors.forename: Marcello André
authors.initials: MA
authors.lastname: Barcinski
mesh_terms: D000818:Animals; D017209:Apoptosis; D004064:Digestive System / Q000166:cytology / Q000469:parasitology / Q000648:ultrastructure; D020287:In Situ Nick-End Labeling; D007894:Leishmania mexicana / Q000166:cytology* / Q000254:growth & development* / Q000472:pathogenicity / Q000648:ultrastructure; D007896:Leishmaniasis / Q000469:parasitology* / Q000473:pathology*; D008018:Life Cycle Stages; D051379:Mice; D010718:Phosphatidylserines / Q000378:metabolism; D011576:Psychodidae / Q000166:cytology / Q000469:parasitology / Q000648:ultrastructure
chemical_list: D010718:Phosphatidylserines