Camellia sinensis solvent extract, epigallocatechin gallate and caffeine confer trophocidal and cysticidal effects against Acanthamoeba castellanii.

We examined the anti-acanthamoebic efficacy of green tea Camellia sinensis solvent extract (SE) or its chemical constituents against Acanthamoeba castellanii by using anti-trophozoite, anti-encystation, and anti-excystation assays. C. sinensis SE (625-5000 µg/mL) inhibited trophozoite replication within 24-72 h. C. sinensis SE exhibited a dose-dependent inhibition of encystation, with a marked cysticidal activity at 2500-5000 µg/mL. Two constituents of C. sinensis, namely epigallocatechin-3-gallate and caffeine, at 100 µM and 200 µM respectively, significantly inhibited both trophozoite replication and encystation. Cytotoxicity analysis showed that 156.25-2500 µg/mL of SE was not toxic to human corneal epithelial cells, while up to 625 µg/mL was not toxic to Madin-Darby canine kidney cells. This study shows the anti-acanthamoebic potential of C. sinensis SE against A. castellanii trophozoites and cysts. Pre-clinical studies are required to elucidate the in vivo efficacy and safety of C. sinensis SE.

Illuminating Host-Parasite Interaction at the Cellular and Subcellular Levels with Infrared Microspectroscopy.

Toxoplasma gondii (T. gondii) is an opportunistic protozoan that can cause brain infection and other serious health consequences in immuno-compromised individuals. This parasite has a remarkable ability to cross biological barriers and exploit the host cell microenvironment to support its own survival and growth. Recent advances in label-free spectroscopic imaging techniques have made it possible to study biological systems at a high spatial resolution. In this study, we used conventional Fourier-transform infrared (FTIR) microspectroscopy and synchrotron-based FTIR microspectroscopy to analyze the chemical changes that are associated with infection of human brain microvascular endothelial cells (hBMECs) by T. gondii (RH) tachyzoites. Both FTIR microspectroscopic methods showed utility in revealing the chemical alterations in the infected hBMECs. Using a ZnS hemisphere device, to increase the numerical aperture, and the synchrotron source to increase the brightness, we obtained spatially resolved spectra from within a single cell. The spectra extracted from the nucleus and cytosol containing the tachyzoites were clearly distinguished. RNA sequencing analysis of T. gondii-infected and uninfected hBMECs revealed significant changes in the expression of host cell genes and pathways in response to T. gondii infection. These FTIR spectroscopic and transcriptomic findings provide significant insight into the molecular changes that occur in hBMECs during T. gondii infection.

Transcriptome Profiling of Toxoplasma gondii-Infected Human Cerebromicrovascular Endothelial Cell Response to Treatment with Monensin.

Central to the progression of cerebral toxoplasmosis is the interaction of Toxoplasma gondii with the blood-brain barrier (BBB) endothelial cells. In the present work, we tested the hypothesis that inhibition of Wnt pathway signalling by the monovalent ionophore monensin reduces the growth of T. gondii infecting human brain microvascular endothelial cells (hBMECs) or microglial cells. The anti-parasitic effect of monensin (a Wnt signalling inhibitor) on the in vitro growth of T. gondii tachyzoites was investigated using two methods (Sulforhodamine B staining and microscopic parasite counting). The monensin inhibited T. gondii growth (50% inhibitory concentration [IC50] = 0.61 µM) with a selective index = 8.48 when tested against hBMECs (50% cytotoxic concentration [CC50] = 5.17 µM). However, IC50 of monensin was 4.13 µM with a SI = 13.82 when tested against microglia cells (CC50 = 57.08 µM), suggesting less sensitivity of microglia cells to monensin treatment. The effect of T. gondii on the integrity of the BBB was assessed by the transendothelial electrical resistance (TEER) assay using an in vitro human BBB model. The results showed that T. gondii infection significantly decreased hBMECs' TEER resistance, which was rescued when cells were treated with 0.1 µM monensin, probably due to the anti-parasitic activity of monensin. We also investigated the host-targeted effects of 0.1 µM monensin on global gene expression in hBMECs with or without T. gondii infection. Treatment of hBMECs with monensin did not significantly influence the expression of genes involved in the Wnt signalling pathway, suggesting that although inhibition of the Wnt signalling pathway did not play a significant role in T. gondii infection of hBMECs, monensin was still effective in limiting the growth of T. gondii. On the contrary, monensin treatment downregulated pathways related to steroids, cholesterol and protein biosynthesis and their transport between endoplasmic reticulum and Golgi apparatus, and deregulated pathways related to cell cycle and DNA synthesis and repair mechanisms. These results provide new insight into the host-modulatory effect of monensin during T. gondii infection, which merits further investigation.