investigating-the-phagocytosis-of-leishmania-using-confocal-microscopy

Investigating the Phagocytosis of <em>Leishmania</em> using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Gonçalo Moniz Institute

CBA mouse macrophages control  infection yet are permissive to . Few studies have been conducted to assess the role played by autophagy in  infection. Therefore, we assessed whether the autophagic response of infected macrophages may account for the differential behavior of these two parasite strains. After 24 h of infection, the LC3-II/Act ratio increased in both - and -infected macrophages compared to uninfected controls, but less than in chloroquine-treated cells. This suggests that  and  activate autophagy in infected macrophages, without altering the autophagic flux. Furthermore, infected cells exhibited higher percentages of DQ-BSA-labeled parasitophorous vacuoles (50%) than those infected by  (25%). However, - and -induced parasitophorous vacuoles accumulated LysoTracker similarly, indicating that the acidity in both compartment was equivalent. At as early as 30 min, endogenous LC3 was recruited to both - and -induced parasitophorous vacuoles, while after 24 h a greater percentage of LC3 positive vacuoles was observed in -infected cells (42.36%) compared to those infected by  (18.10%). Noteworthy, principal component analysis (PCA) and an hierarchical cluster analysis completely discriminated -infected macrophages from -infected cells accordingly to infection intensity and autophagic features of parasite-induced vacuoles. Then, we evaluated whether the modulation of autophagy exerted an influence on parasite infection in macrophages. No significant changes were observed in both infection rate or parasite load in macrophages treated with the autophagic inhibitors wortmannin, chloroquine or VPS34-IN1, as well as with the autophagic inducers rapamycin or physiological starvation, in comparison to untreated control cells. Interestingly, both autophagic inducers enhanced intracellular  and  viability, while the pharmacological inhibition of autophagy exerted no effects on intracellular parasite viability. We also demonstrated that autophagy induction reduced NO production by - and -infected macrophages but not alters arginase activity. These findings provide evidence that although -induced parasitophorous vacuoles recruit LC3 more markedly,  and  similarly activate the autophagic pathway in CBA macrophages. Interestingly, the exogenous induction of autophagy favors  intracellular viability to a greater extent than  related to a reduction in the levels of NO.

Autophagic Induction Greatly Enhances  Intracellular Survival Compared to  in CBA/j-Infected Macrophages.

Zika virus (ZIKV), a member of the Flaviviridae family, was brought into the spotlight due to its widespread and increased pathogenicity, including Guillain-Barré syndrome and microcephaly. Neural progenitor cells (NPCs), which are multipotent cells capable of differentiating into the major neural phenotypes, are very susceptible to ZIKV infection. Given the complications of ZIKV infection and potential harm to public health, effective treatment options are urgently needed. Betulinic acid (BA), an abundant terpenoid of the lupane group, displays several biological activities, including neuroprotective effects. Here we demonstrate that Sox2 NPCs, which are highly susceptible to ZIKV when compared to their neuronal counterparts, are protected against ZIKV-induced cell death when treated with BA. Similarly, the population of Sox2 and Casp3 NPCs found in ZIKV-infected cerebral organoids was significantly higher in the presence of BA than in untreated controls. Moreover, well-preserved structures were found in BA-treated organoids in contrast to ZIKV-infected controls. Bioinformatics analysis indicated Akt pathway activation by BA treatment. This was confirmed by phosphorylated Akt analysis, both in BA-treated NPCs and brain organoids, as shown by immunoblotting and immunofluorescence analyses, respectively. Taken together, these data suggest a neuroprotective role of BA in ZIKV-infected NPCs.

Betulinic Acid Exerts Cytoprotective Activity on Zika Virus-Infected Neural Progenitor Cells.

The heat shock protein 90 (Hsp90) is thought to be an excellent drug target against parasitic diseases. The leishmanicidal effect of an Hsp90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), was previously demonstrated in both in vitro and in vivo models of cutaneous leishmaniasis. Parasite death was shown to occur in association with severe ultrastructural alterations in , suggestive of autophagic activation. We hypothesized that 17-AAG treatment results in the abnormal activation of the autophagic pathway, leading to parasite death. To elucidate this process, experiments were performed using transgenic parasites with GFP-ATG8-labelled autophagosomes. Mutant parasites treated with 17-AAG exhibited autophagosomes that did not entrap cargo, such as glycosomes, or fuse with lysosomes. ATG5-knockout (Δ) parasites, which are incapable of forming autophagosomes, demonstrated lower sensitivity to 17-AAG-induced cell death when compared to wild-type (WT) , further supporting the role of autophagy in 17-AAG-induced cell death. In addition, Hsp90 inhibition resulted in greater accumulation of ubiquitylated proteins in both WT- and Δ-treated parasites compared to controls, in the absence of proteasome overload. In conjunction with previously described ultrastructural alterations, herein we present evidence that treatment with 17-AAG causes abnormal activation of the autophagic pathway, resulting in the formation of immature autophagosomes and, consequently, incidental parasite death.

Article	Year
Autophagic Induction Greatly Enhances Intracellular Survival Compared to in CBA/j-Infected Macrophages. Frontiers in microbiology\| PubMed ID: 30158914	2018
Betulinic Acid Exerts Cytoprotective Activity on Zika Virus-Infected Neural Progenitor Cells. Frontiers in cellular and infection microbiology\| PubMed ID: 33251156	2020
17-AAG-Induced Activation of the Autophagic Pathway in Is Associated with Parasite Death. Microorganisms\| PubMed ID: 34069389	2021

Beatriz R. S. Dias

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