Leishmania majorparasite stage‐dependent host cell invasion and immune evasion

Abstract

Leishmania pathogenesis is primarily studied using the disease-inducing promastigote stage of Leishmania major. Despite many efforts, all attempts so far have failed to culture the disease-relevant multiplying amastigote stage of L. major. Here, we established a stably growing axenic L. major amastigote culture system that was characterized genetically, morphologically, and by stage-specific DsRed protein expression. We found parasite stage-specific disease development in resistant C57BL/6 mice. Human neutrophils, as first host cells for promastigotes, do not take up amastigotes. In human macrophages, we observed an amastigote-specific complement receptor 3-mediated, endocytotic entry mechanism, whereas promastigotes are taken up by complement receptor 1-mediated phagocytosis. Promastigote infection of macrophages induced the inflammatory mediators TNF, CCL3, and CCL4, whereas amastigote infection was silent and resulted in significantly increased parasite numbers: from 7.1 ± 1.4 (after 3 h) to 20.1 ± 7.9 parasites/cell (after 96 h). Our study identifies Leishmania stage-specific disease development, host cell preference, entry mechanism, and immune evasion. Since the amastigote stage is the disease-propagating form found in the infected mammalian host, the newly developed L. major axenic cultures will serve as an important tool in better understanding the amastigote-driven immune response in leishmaniasis.

Apoptotic-likeLeishmaniaexploit the host´s autophagy machinery to reduce T-cell-mediated parasite elimination

“…61,62 Apoptotic and viable promastigotes were purified from stationary phase promastigotes by magnetic separation, as described previously. 1 …”

“…Cells were permeabilized by saponin (Sigma, S7900) and stained with DAPI, a polyclonal rabbit anti-human LC3 Ab (Cell Signaling Technology, 2775S), and an anti-Lm polyclonal Ab obtained from Lm infected Balb/c mice. 62 For detection, chicken antimouse Alexa 488 (A21200) and goat anti-rabbit Alexa Fluor 568 (A11036) were used (Molecular Probes/Invitrogen). Chamber slides were analyzed using a Zeiss Observer, Zeiss LSM7 Live and Axiovision/ZEN 2009 Software (Carl Zeiss, G€ ottingen, Germany).…”

“…62 To determine the apoptotic rate of the promastigotes populations, parasites were stained using ANXA5 Alexa Fluor 647 (Molecular Probes/Invitrogen, A23204). For FACS analysis of human cells, 0.3 £ 10 6 cells were labeled with mouse anti-human CD3-PE (556612), CD4-PE (555347), or CD8-pacific blue (558207), all purchased from BD Bioscience and the additional isotype controls as defined by the manufacturer.…”

Leishmania amazonensis Amastigotes Trigger Neutrophil Activation but Resist Neutrophil Microbicidal Mechanisms

“…Specifically, the newly described method for culturing axenic L. major amastigotes yields parasites that are not readily internalized by human neutrophils (60). Additionally, it has been shown that L. donovani amastigotes derived from infected hamsters are degraded by human neutrophils (24).…”

Leishmania Promastigotes Induce Cytokine Secretion in Macrophages through the Degradation of Synaptotagmin XI

“…Indeed, we recently reported that Syt XI, which inhibits vesicle fusion (38), dampens the secretion of both TNF and IL-6 (26). Interestingly, it has been demonstrated that L. major promastigotes induce the secretion of TNF and IL-6 following their internalization by macrophages (9,(39)(40)(41)(42). This raises the possibility that these parasites could disrupt membrane fusion regulators to induce proinflammatory cytokine release.…”