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 (PSPOS) sub-population was confirmed by TUNEL staining and DNA laddering. Transmission electron microscopy revealed morphological alterations in PSPOS metacyclics such as DNA condensation, cytoplasm degradation and mitochondrion and kinetoplast destruction, both in in vitro cultures and in sand fly guts. TUNELPOS 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 PSPOS and PS-negative (PSNEG) parasites were simultaneously added to the cell culture or inoculated in the mammalian host. The viable PSNEG promastigote was the infective form, as shown by following the fate of fluorescently labeled parasites, while the PSPOS 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.
Highlights
Programmed cell death by apoptosis plays a central role in normal tissue development and homeostasis
A sub-population of metacyclic promastigotes from in vitro cultures or from the sand fly gut displays PS on its surface To compare the amount of PS exposure by logarithmic and stationary-phase promastigotes, parasites obtained from 2 day-old cultures after at least 3 short-term consecutive passages, and from 6 to 7 day-old cultures, as well as a population enriched for infective metacyclic forms, were assessed for PS exposure after annexin V (AnV) binding
To assess if exposed PS might play a role in natural infections, we looked for AnV binding in metacyclic promastigotes purified from dissected guts of Lutzomyia longipalpis at 5 and 9 days postinfection (p. i.)
Summary
Programmed cell death by apoptosis plays a central role in normal tissue development and homeostasis. PSPOS population displays ultrastructural and biochemical features of apoptotic death, while the PSNEG population is the truly infective one Both forms, when purified, are able to internalize into macrophages; intracellular multiplication of PSNEG forms only occurs when the phagocytes are infected in the presence of PSPOS forms, which are capable of inhibiting production of nitric oxide by activated macrophages. The cell biology of PS exposure in promastigotes, which occurs via apoptotic death, differs from the one in amastigotes occurring via apoptotic mimicry [12] They take place at different stages of the disease caused by La: the first one is required at the moment of infection, and the second one, for disease progression in the mammalian host.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
